Neuropeptide S (NPS) is a neuropeptide with cellular actions in arousal and anxiety-related nuclei: Functional implications for effects of NPS on wakefulness and mood

Accepted Manuscript Neuropeptide S (NPS) is a neuropeptide with cellular actions in arousal and anxietyrelated nuclei: Functional implications for effects of NPS on wakefulness and mood Vincenzo Roncacè, Filip Souza Polli, Minella Zojicic, Kristi Anne Kohlmeier PII:

S0028-3908(17)30298-8

DOI:

10.1016/j.neuropharm.2017.06.025

Reference:

NP 6761

To appear in:

Neuropharmacology

Received Date: 7 March 2017 Revised Date:

2 June 2017

Accepted Date: 23 June 2017

Please cite this article as: Roncacè, V., Polli, F.S., Zojicic, M., Kohlmeier, K.A., Neuropeptide S (NPS) is a neuropeptide with cellular actions in arousal and anxiety-related nuclei: Functional implications for effects of NPS on wakefulness and mood, Neuropharmacology (2017), doi: 10.1016/ j.neuropharm.2017.06.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Neuropeptide S (NPS) Is a Neuropeptide with Cellular Actions in Arousal and Anxiety-Related Nuclei: Functional Implications for effects of NPS on Wakefulness and Mood Vincenzo Roncacè#, Filip Souza Polli, Minella Zojicic and Kristi Anne Kohlmeier*

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Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 160, 2100 Copenhagen Ø, Denmark

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Text pages: 33 (excluding references) Figures: 9 Table: 0

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Running title: NPS has cellular actions on DR and LDT neurons

Acknowledgements: The authors wish to acknowledge Tina Axen, who provided technical assistance with the immunohistochemical studies presented in this work. KAK would like to thank Lisiane Souza for providing the inspiration for this study.

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VR was supported by a Marco Polo Fellowship (Dept Life Quality Studies, University of Bologna) and a research fellowship (University of Bologna). FSP is supported by a PhD stipend from CNPq (Brazil). Experiments funded by a grant to KAK (Philip Morris External Research Program, USA), and university support (Drug Design and Pharmacology, University of Copenhagen, Denmark). The authors would like to state that they have no conflict of interest with the work presented in this manuscript.

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* Author to whom correspondence should be addressed: Dr. Kristi Anne Kohlmeier Department of Drug Design and Pharmacology University of Copenhagen Jagtvej 160 2100 Copenhagen, Denmark [email protected] Present Address: Dr. Vincenzo Roncacè Department for Life Quality Studies School of Pharmacy, Biotechnology and Sport Science University of Bologna

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ACCEPTED MANUSCRIPT ABSTRACT

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Neuropeptide S (NPS) is a peptide recently recognized to be present in the CNS, and believed to play a role in vigilance and mood control, as behavioral studies have shown it promotes arousal and has an anxiolytic effect. Although NPS precursor is found in very few neurons, NPS positive fibers are present throughout the brain stem. Given the behavioral actions of this peptide and the wide innervation pattern, we examined the cellular effects of NPS within two brain stem nuclei known to play a critical role in anxiety and arousal: the dorsal raphe (DR) and laterodorsal tegmentum (LDT). In mouse brain slices, NPS increased cytoplasmic levels of calcium in DR and LDT cells. Calcium rises were independent of action potential generation, reduced by low extracellular levels of calcium, attenuated by IP3 - and ryanodine (RyR)-dependent intracellular calcium store depletion, and eliminated by the receptor (NPSR) selective antagonist, SHA 68. NPS also exerted an effect on the membrane of DR and LDT cells inducing inward and outward currents, which were driven by an increase in conductance, and eliminated by SHA 68. Membrane actions of NPS were found to be dependent on store-mediated calcium as depletion of IP3 and RyR stores eliminated NPS-induced currents. Finally, NPS also had actions on synaptic events, suggesting facilitation of glutamatergic and GABAergic presynaptic transmission. When taken together, actions of NPS influenced the excitability of DR and LDT neurons, which could play a role in the anxiolytic and arousal-promoting effects of this peptide.

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ACCEPTED MANUSCRIPT INTRODUCTION

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Reverse pharmacology successfully identified neuropeptide S (NPS), which is a 20 amino acid protein that selectively binds to, and activates, an orphan G-protein coupled receptor called NPSR (Xu et al., 2004). Although it is one of the least abundant neuropeptides recognized to be present in the brain, this protein and its precursors can be found in all vertebrates except fish (Adori et al., 2015; Reinscheid, 2007). Since the recognition of this peptide system, the distribution of NPS and NPSR gene expression has been mapped in detail across the rodent brain by in-situ hybridization (Xu et al., 2007, 2004). Although NPS-positive fibers were found distributed throughout the brain, NPS precursor was only found in the Kölliker-Fuse nucleus and in a collection of neurons near the noradrenergic locus coeruleus (LC) (Clark et al., 2011). This extremely restricted pattern of NPS precursor is in contrast to the much broader presence of NPSR mRNA which is found throughout the brain, consistent with extensive NPS innervation of target nuclei via detected NPS-positive fibers (Clark et al., 2011).

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Since the discovery of the NPS peptide system in 2004, several laboratories have investigated the behavioral effects of NPS. In vivo paradigms designed to evaluate anxiety, such as open field testing, elevated plus or zero maze, light-dark box preference, four-plate test, stress-induced hyperthermia, social fear conditioning, and social defeat, have shown that central administration of NPS in both rats and mice produces an anxiolytic effect (Leonard et al., 2008; Lukas and Neumann, 2012; Pulga et al., 2012; Rizzi et al., 2008; Xu et al., 2004; Zoicas et al., 2016). In mice, intra-amygdala administration of NPS resulted in an acute reduction of general anxiety as measured in open field and elevated plus maze experiments. Furthermore, the same treatment resulted in accelerated extinction of conditioned fear evidenced by a reduction of freezing behavior in an auditory fear conditioning paradigm (Jüngling et al., 2008). In addition to anxiolytic actions, the NPS system exerts profound arousal enhancing effects as the i.c.v. administration of NPS in rats reduced all stages of sleep and promoted arousal even at low doses (Xu et al., 2004), and treatment with an NPS analogue reduced the duration of ketamine-induced anesthesia (Kushikata et al., 2011). Consistent with a role of NPS in reducing anxiety and in promoting arousal, inhibition of the NPS system by use of NPSR antagonists has been shown to exert opposite effects, including exacerbation of anxious-like behaviors and reductions in durations of aroused states. For example, injection of a NPS non-peptidergic NPSR antagonist, SHA 68, locally into the amygdala enhanced the freezing response to conditioned stimuli (Jüngling et al., 2008). Central administration of the NPSR antagonist, [D-Cys(tBu) (5)] NPS, in rats decreased the animals wakefulness state, and significantly increased the amount of non-rapid eye movement sleep (Oishi et al., 2014). Although NPS actions have not been characterized pharmacologically in humans, intriguingly, genetic studies have suggested this peptide could play a role in human behaviors involving anxiety and arousal. Genomic analysis revealed an association of the single nucleotide polymorphism (SNP), rs324981, lying at triplet position 107 of the NPSR1 gene, with sensitivity to anxiety and panic disorder, suggesting a relationship of this genetic difference to the pathogenesis of amygdala-based, fear-related disorders (Dannlowski et al., 2011). The SNP rs324981 was also linked to a significant reduction in the total duration of sleep and the amount of rest in individuals with the homozygote genotype, suggesting this variant of the NPSR1 was involved in alterations of control of arousal state (Spada et al., 2014). These effects were likely to extend to actions of the NPS system in non-amygdalar regions of the brain involved in sleep and arousal control, such as the hypothalamus and brain stem.

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Taken together, findings from behavioral studies in rodents and genetic studies in humans strongly suggest that NPS reduces anxiety and promotes arousal, however, the cellular effects of this peptide have not been widely examined, although a few reports do exist. Isolated cell culture studies have shown that activation of the NPS1R generates rises in calcium via activation of the Gq and/or Gs pathways (Bernier et al., 2006; Erdmann et al., 2015; Gupte et al., 2004; Liao et al., 2016; Reinscheid et al., 2005; Tancredi et al., 2007; Xu et al., 2004). Studies of native neurons in brain slices revealed that NPS activated glutamatergic transmission in the amygdala, which in turn modulated GABAergic synaptic activity (Jüngling et al., 2008; Meis et al., 2011, 2008; Zhang et al., 2016). Within the hypothalamus, NPS modulates an ionic conductance which underlies a biphasic effect of membrane depolarization and hyperpolarization (Yoshida et al., 2010). And relevant to mechanisms underlying its arousal-promoting actions, NPS-positive cells adjacent to the LC were depolarized by corticotropin releasing factor (CRF) acting at the CRF-1 receptor to an extent which was sufficient to induce cell firing, indicating that stress-mediated activation of the NPS system within the extended LC could act as part of the arousal response to stressor stimuli (Jüngling et al., 2012).

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However, while the amygdala, hypothalamus and stress-related signaling in the LC are involved in mediating anxiety and arousal, these regions do not represent the only nuclei in the brain controlling such behaviors. Accordingly, if we are to fully understand the role that NPS plays in human behaviors, further studies of the cellular actions of this peptide are needed. To this end, we examined the cellular effects of NPS within two brain stem nuclei known to play a role in anxiety and arousal: the dorsal raphe (DR) and laterodorsal tegmentum (LDT). The DR, located in the rostral pontine and caudal midbrain tegmentum, contains the largest group of serotonergic neurons within the brain (Vertes and Crane, 1997). Via a diffuse serotonin-containing projection system throughout the brain, the DR has been shown to play a key role in controlling physiologic functions like: sleep-wake cycles (Monti, 2010; Sakai, 2011), emotional behavior, including anxiety and motivated behavioral states (Teissier et al., 2015). The LDT is located in the dorsolateral pontine reticular formation and is an integral player in the ascending reticular activating system (Mena-Segovia, 2016; Mesulam et al., 1983; Moruzzi and Magoun, 1995). Via a diffuse rostral and caudal projection system, cholinergic, glutamatergic and GABAergic LDT neurons play a role in generation of the phenomenon of rapid eye movement sleep, control of the sleep-wake cycle, modulation of attention and induction of arousal (for review, see Kohlmeier et al., 2013; Moruzzi and Magoun, 1995; Steriade, 1999; Van Dort et al., 2015). Using a combination of calcium imaging, whole cell electrophysiology and immunohistochemistry, we present data which show for the first time that NPS has strong cellular actions on both pre and postsynaptic neurons of the DR and LDT which involve alterations in membrane currents, synaptic activity and intracellular calcium, that were specific to activation of the NPSR. The ability of NPS to influence cellular excitability of DR and LDT neurons could be expected to alter output from these neuronal groups to target regions; and thereby, play a role in behaviors controlled by these two nuclei, including regulation of mood, anxiety, the sleep-wake cycle, attention and arousal. Findings from our cellular studies are expected to contribute to future develop of novel pharmacotherapies based on NPS, which could provide a more tailored and successful management of anxiety and disorders presenting with abnormal arousal.

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ACCEPTED MANUSCRIPT METHODS Animals and brain slice preparation Brain slices containing the DR or the LDT were collected from NMRI wild-type mice (Harlan Mice laboratories, Denmark) between the postnatal ages of 8–21 days. Mice were received in a litter containing 10 pups, with a foster mother, and housed in a temperature controlled room (22–23 °C) with a 12:12 h light-dark cycle. Tap water and laboratory chow were available ad libitum.

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To obtain brain slices, the animal was deeply anesthetized with isoflurane, and following loss of the righting reflex and failure to respond to a paw pinch, the animal was decapitated and a block of the brain containing the DR and LDT was removed from the cranial box. Following further blocking, the remaining tissue was sliced in ice-cold artificial cerebrospinal fluid (ACSF) into 250 µm coronal brain slices which contained the DR or the LDT using a Leica vibrotome (VT 1200S, Leica, Germany) which had been calibrated using Vibrocheck (Leica) so as to reduce potentially damaging vertical deflection, thereby minimizing tissue damage. Following slicing, the tissue was incubated in 95% O2 -5% CO2 (carbogen) saturated ACSF for 15 min at 37 °C, and then left to equilibrate to room temperature for 1 hour prior to recordings. Following equilibration, slices were moved to the recording chamber, and continuously perfused with ACSF at room temperature at a flow rate of 3 mL/min.

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External and Pipette Solutions The majority of the experiments were performed in ACSF containing in mM: NaCl 124; KCl 5; Na2HPO4 1.12; CaCl2*2H2O 2.7; MgSO4 (anhydrous) 1.12; Dextrose 10; NaHCO3 26; saturated with carbogen. In recordings designed to examine the role of flux of calcium across the membrane, a low calcium external solution was utilized which was shown in preliminary studies to inhibit calcium entry across the membrane of DR and LDT cells sufficient to eliminate action potentials and ongoing excitatory and inhibitory phasic synaptic activity. The low calcium solution contained in mM: NaCl 127; KCl 5; Na2HPO4 1.2; CaCl2*2H2O 2.7, MgSO4 (anhydrous) 1.2; Dextrose 10; NaHCO3 26. It should be noted that solutions absent of calcium were not utilized as previous experience has shown that long term recordings, which were required in the present study, can not be sustained in the DR or LDT in zero or highly buffered calcium conditions. In recordings in which we wished to shift the reversal potential of potassium in order to implicate a potassium conductance in phenomenon seen, a high potassium-containing ACSF was used, which contained in mM: NaCl 124; KCl 12.62; Na2HPO4 1.12; CaCl2*2H2O 2.7; MgSO4 (anhydrous) 1.12; Dextrose 10; NaHCO3 26. For the majority of patch clamp recordings, pipettes were filled with a solution containing in mM: Kgluconate 144; KCl 2; HEPES 10; EGTA (tetraacetic acid) 0.2; Mg-ATP 5 and Na-GTP 0.3. For patch clamp recording of the inhibitory postsynaptic currents, the driving force for chloride was altered by enriching the pipette solution with chloride anion by using a pipette solution containing in mM: KCl 144; EGTA 0.2; MgCl2 3; HEPES 10; NaGTP 0.3; Na2ATP 4. For calcium imaging recordings, EGTA in the patch solution was substituted with 25 µM of bis-Fura 2 (Molecular Probes). For a post hoc recovery of cells in order to conduct immunohistochemistry so as to identify the neurotransmitter content of the recorded neurons, in all recordings, biotinylated Alexa-594 (25 µM, Molecular Probes)

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ACCEPTED MANUSCRIPT was also included in the pipette, which passively diffused into the internal cytoplasm permitting post hoc identification of cells from which recordings had sourced.

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Patch Clamp Recordings Patch pipette electrodes of thin wall borosilicate glass were produced using a Sutter P-97 horizontal puller (Sutter Instruments, USA) and once filled with internal solution, exhibited resistances between 6–10 MΩ . Under visual guidance using differential interference contrast optics on an upright microscope (Olympus BX50WI, Germany), neurons in the DR and LDT were visualized with a 40× water immersion objective (NA 0.8, Olympus). High-resistance seals (> 1 mΩ) were established between the patch pipette and the cell membrane via a patch clamp EPC9 amplifier (HEKA, Germany) in voltage clamp mode guided by Pulse (HEKA; version 9.0). After that time, seals were broken by membrane rupture, and a holding current sufficient to maintain, or clamp, the cell at a voltage of -60 mV was applied. Recordings of currents were sampled at a rate of 10 kHz using AxoScope 10.2 (Molecular Devices Corporation, USA) and an Axon Digidata 1440A digitizer (Molecular Devices Corporation). Recordings were discarded if the current necessary to hold the cell at -60 mV exceeded 50 pA or if the series resistance exceeded 30 MΩ. It should be noted that the liquid junction potential was calculated to be ~ 13 mV using gluconate-containing patch solution, but data were not corrected for this voltage.

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Both the DR and the LDT are heterogenous nuclei, comprised of the principal serotonergic and cholinergic, respectively, neurons, but also of other cell types. To maximize the probability of including all of the cell types in our examination of NPS effects, a variety of cell shapes was selected for patch clamp recordings. Serotonergic or cholinergic neuronal phenotype was definitively determined by use of immunohistochemistry post-hoc. To maintain consistency in the recordings, the dorsal portion of the dorsal raphe, which is rich in serotonergic neurons, and that receives projections from areas associated in control of emotional behavior (Van Bockstaele et al., 1993; Didier-Bazes et al., 1997; Commons et al., 2003), was utilised. For LDT recordings, the medial portion of the LDT in which cholinergic neurons are at their highest density, was the anatomical region from which cells were selected (Boucetta and Jones, 2009; Boucetta et al., 2014).

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In some recordings, current-voltage (IV) relationships were determined in order to elucidate the reversal potential of the ionic conductances activated by NPS. Accordingly, in voltage clamp mode, a voltage step protocol from the starting holding voltage of -60 mV with a delta increment of 10 mV (400 ms step duration) was applied in order to generate input-output curves. Tetrodotoxin (TTX) was present in these recordings to block action potential generation. Reversal potentials of the drug-elicited currents, which were suggestive of the ionic conductance(s) underlying the currents, were determined from the crossing of the control curve with that of the drug-induced curve. In order to examine whether NPS cellular actions resulted in the functional outcome of altering firing, we monitored the frequency of action potentials in current clamp mode before and after exposure to the peptide. In this configuration, current was applied to depolarize the neuron to -45 mV, which was sufficient to induce a sustained spontaneous firing of action potentials. The intervals of action potentials were measured and averaged during a 30 sec epoch before and a 30 sec epoch after NPS.

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Drugs NPS (Sigma) was diluted in ddH2O at stock concentrations and stored in aliquots at -20 °C till usage, at which time, the stock solution was diluted in ACSF to a final concentration of either 100 nM for bath application conducted during bolus loaded, multiple cell, calcium imaging experiments or 10 µM for pressure application with a picospritzer (20 PSI, 500 ms duration) for patch clamp and single neuron calcium imaging. The concentration of NPS selected for bath application (100 nM) was similar, albeit a bit reduced, from that used by other authors (200 nM; Meis et al., 2008). The concentration used in our study for picospritizing was higher than that used in other studies with the same method of application (5 µM; Meis et al., 2008); however, direct comparisons are a bit challenging as other authors used a lower pressure (5 psi) for a longer duration (10-20 s). In preliminary experiments, we found that 10 µM applied with a psi of 20 and a duration of 500 ms gave reliable and detectable responses. To inhibit voltage-operated sodium channels underlying action potential firing, tetrodotoxin (TTX; Tocris) was added to the ACSF at a final concentration of 500 nM.

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To block the NPS receptor, we used the well-validated NPS receptor antagonist, SHA 68 (Tocris) and selected a concentration utilized in other studies which was shown to be effective at inhibiting NPS responses (Okamura et al., 2008). Accordingly, stock solutions of SHA 68 were prepared as previously reported by dilution in 50 mM DMSO/Ethanol (1:1). Final concentrations were diluted in ACSF containing 0.1% of serum bovine albumin (BSA) to 10 µM (final ratio 1/1:10000 DMSO/Ethanol:ACSF) or to 100 µM (final ratio 1/1:1000 DMSO/Ethanol:ACSF). Solvents at these dilutions were shown to have no actions on DR and LDT neurons (data not shown).

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In recordings designed to examine effects of NPS on inhibitory synaptic currents (IPSCs), excitatory synaptic currents (EPSCs) due to glutamatergic actions on AMPA, Kainate and NMDA receptors were blocked by the ionotropic glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX; 15 µM) and (2R)-amino-5-phosphonopentanoate (AP5; 50 µM), which were added to the ACSF during the recording of IPSCs with the high chloride, pipette recording solution. While the majority of recordings designed to examine the effect of NPS on downward going EPSCs were conducted in the absence of blockade of IPSCs (as these were upward going deflections), confirmation of identical effects of NPS on EPSCs in presence of strychnine (2.5 µM; Sigma) and SR-95531 (gabazine, 10 µM, Sigma) to block glycinergic and GABAergic inhibitory synaptic currents and CGP 55845 (10 µM, Tocris) to block GABAB receptor-mediated responses, was obtained in a subpopulation of cells. Examination of effects of NPS on firing frequency were conducted in the presence of a cocktail of these glutamate, glycine and GABA receptor blockers, and findings were compared to those in absence of these blockers. To inhibit IP3-mediated, calcium pumps on the sarcoplasmic reticulum, and thereby ‘dump’ this intracellular store of calcium, cyclopiazonic acid (CPA; Sigma) was used, which was prepared in 10 mM stock of DMSO, and diluted in ACSF to a final concentration of 10 µM (final ratio 1:1000 DMSO/ACSF). To deplete the ryanodine receptor (RyR)-mediated calcium stores, caffeine diluted in ACSF to a final concentration of 20 mM, was applied. Success of these two compounds in releasing intracellular calcium are shown as an insert in Figure 8. Calcium imaging

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ACCEPTED MANUSCRIPT In the present study, two types of single-photon, calcium imaging, both relying on the ratiometric, BAPTA-based molecule Fura 2 were conducted. As an initial screen to determine whether NPS had actions on activity of DR and LDT neurons, multiple-cell loading of the cell membrane-permeant form of Fura, Fura 2-AM, was utilized. For a more in-depth investigation of calcium dynamics stimulated by NPS, single-cell imaging was conducted in which the cell membrane-impermeant form of Fura, bisFura 2, passively diffused into the cell via the patch pipette.

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For multiple-cell loading, brain slices containing the DR or LDT were incubated at 31 °C in Fura 2AM (15 µM in DMSO/ACSF; Molecular Probes, Invitrogen, Denmark) which had been saturated with carbogen for 10 minutes. The loading time was increased by 1 minute for every day of age greater than postnatal day 10, that we and others (MacLean and Yuste, 2009; McNair and Kohlmeier, 2015) have shown enhances loading quality, which generally reduces as the postnatal age of the tissue increases. Following incubation, slices were transferred to the recording chamber where they were rinsed with ACSF for 30 minutes in order to wash out any unincorporated calcium imaging dye prior to data collection.

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For both types of calcium imaging, changes in fluorescent emissions of Fura, which are used as indirect indicators of changes in calcium, were detected using a cooled, 12 bit CCD fluorescent camera (Sensicam, PCO Instruments, Germany) with Xenon illumination. The camera and shutter were controlled by an ICU operated by TILL-VISION software (Till Photonics, Germany). The camera was fixed on the head stage of the upright microscope. The DR and LDT were identified by wellestablished landmarks under 4× optics, and under higher-magnification, individual cells were noted, and delineated by regions of interest (ROIs). Fura is a ratiometric dye which if viewed across two wavelengths, allows for minimization of measurement errors due to dye bleaching following exposure and to dye leakage. Accordingly, changes in fluorescence were measured within the ROIs at two excitation wavelengths, 340 nm and 380 nm, at which rises in calcium increase, and decrease, respectively, the fluorescence emission. Fluorescence values at 340 nm and 380 nm were then averaged across the ROI, and, following subtraction of auto fluorescence as determined from imaging in a region devoid of fluorescent cells, were rationed in a dimensionless value of F = F340/F380. Drug-induced changes in fluorescence are reported as DeltaF/F (DF/F), which reflects the maximum change in fluorescence during the drug application (DF) divided by the measure of fluorescence at baseline (F), which was calculated as the average of the ratiometric fluorescence of 10 frames of exposition prior to drug application. For each wavelength, to reduce exposure times so as to limit bleaching of the dye, and phototoxicity, time of exposure was restricted to that which maintained the brightest pixel in the ROIs between 7–10% of the upper limit of the dynamic range (4096 grey levels). In figures, upward going deflections in DF/F indicate rises in intracellular calcium, and cells exhibiting a rise exceeding a 2% change in DF/F were considered as having responded. Data Analysis The amplitudes of the membrane currents elicited by NPS were measured by using Clampfit 10.3 (Molecular Devices, USA), and determined as the difference between 1 ms of current averaged before the NPS application and the average of 1 ms of current at the maximum deflection. Firing frequency was calculated using the instantaneous frequency calculator in Clampfit 10.3 and reported as

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normalized averaged values. Changes in DF/F of smooth spiker and plateaus responses which were categorized based on kinetic criteria previously reported (Kohlmeier et al., 2004), were measured as the difference between the average of 5 frame pairs before NPS and 5 frame pairs at the maximal effect. The amplitude of spiker responses was calculated as the average of the 3 largest spikes. Statistical comparisons were performed using a paired or unpaired Student’s t-test, or when differences in the variance of the population were detected, the non-parametric Mann-Whitney test was used (Graphpad Prism, USA). The Fisher’s exact test was used to compare proportions of cells responding, or response type, across populations (Graphpad Prism). Significance for all these tests was set at 0.05. Spontaneous synaptic events (EPSCs and IPSCs), as well as miniature synaptic events (mEPSCs and mIPSCs), were detected and analyzed using MiniAnalysis (Synaptosoft, USA). Current recordings with epochs of 30 s duration, which is a duration of time within the range of that used in other studies examining effects of NPS on synaptic events (Meis et al., 2008, 2011), were selected for analysis just prior to, and at 360390 s after the application of NPS. Where noted, epochs occurring at a time point exceeding 390 s post peptide application was also analyzed to determine persistence of NPS effects. The pre and post drug 30 s epochs were evaluated for the number of PSC events, the inter-event intervals, and the amplitude of events. Statistical significance of the cumulative distributions of these parameters between control and NPS conditions was determined within the same cell by using the non-parametric statistic, the Kolmogorov-Smirnov (K-S test), with significance set at p values less than 0.05. We first determined whether NPS induced a significant change in the distribution of intervals or amplitudes for each cell. If a cell exhibited a significant change in the distribution of the intervals of the PSCs or of the amplitudes, an average for that cell was calculated for the significant parameter, and this was used to calculate an average and SEM for the population of cells which exhibited a significant response. Results are presented as mean values ± standard error of the mean. The figures were made using Igor Pro software (Wavemetrics, USA) and Prism Graphpad.

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Immunohistochemistry To identify recorded cells as being within the confines of the DR or the LDT, and/or to characterize their phenotype as serotonergic or cholinergic, we used immunohistochemistry for markers of TpH or bNOS, respectively. To this end, following recordings, brain slices were placed in 4% paraformaldehyde for a minimum of four hours and then stored in 30% sucrose in phosphate buffer saline solution for a minimum of 24 hours. Slices were re-sectioned to a thickness of 40 µm on a cryostat (Leica CM 3050S) and incubated in either an anti-tryptophan hydroxlase antibody, or an antibNOS antibody (TpH: rabbit polyclonal to TPH2, cat # ab111828, abcam; bNOS: rabbit polyclonal, cat # N7280, Sigma-Aldrich, Denmark). After incubation overnight at room temperature in the appropriate primary antibody, following extensive rinsing of the slice, a fluorescent secondary antibody was added for 30 minutes (anti-rabbit, goat, cat # A11008, Molecular probes, Denmark). Recorded cells were detected by optics optimized for visualization of the Alexa-594 (560 nm wavelength), which had passively diffused into the cell from the patch pipette. Identification of the Alexa-594 filled cell as serotonergic or cholinergic was done by detection of the co-presence, or absence, of the secondary antibody when viewed under appropriate optics (488 nm wavelength).

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ACCEPTED MANUSCRIPT RESULTS

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NPS promotes increases in calcium in DR and LDT neurons As there are no reports of the presence of NPS related activity in the DR or LDT, but NPS is known in other cell systems to increase cytoplasmic calcium via activation of the NPSR (Bernier et al., 2006; Erdmann et al., 2015; Gupte et al., 2004; Liao et al., 2016; Reinscheid et al., 2005; Tancredi et al., 2007; Xu et al., 2004), our first experiments were focused on quickly determining whether NPS had actions on cells within these two nuclei by using a calcium activity-based assay: bulk-load, Fura 2-AM imaging. With this technique, large numbers of cells within nuclei can be filled with the cell-permeant fluorescent dye, Fura 2-AM, which after it crosses cell membranes, becomes trapped within cells following enzymatic cleavage, where it changes its fluorescent emissions if intracellular calcium alters. Therefore, brain slices containing the DR (n = 39 animals) and the LDT (n = 40 animals) were obtained from mice (P8–P20) and loaded with Fura 2-AM (Fig 1: A1, B1) and changes in fluorescence (DeltaF/F, DF/F) induced by bath application of NPS (3 mL, 100 nM in ACSF) were examined. In preliminary studies, there were no differences across gender, or age as responses obtained in slices from a young group of mice (P8–13) were similar to those obtained in an older group (P14–20), therefore, data were pooled across these parameters. At the end of experiments, brain slices were immunohistochemically processed for TpH, to reveal serotonergic neurons of the DR, or for bNOS, to reveal cholinergic neurons in the LDT, in order to ensure that the brain slices contained the nuclei of interest and that the recordings were conducted in the DR or LDT (Fig 1: A, B, two boxed panels on left).

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Our calcium imaging studies provide the first evidence that NPS has actions on DR and LDT cells. Within the DR, rises in fluorescence of the Fura 2 loaded cells were elicited in 36% of the cells by NPS (n = 104/298), indicating this peptide induces rises in intracellular calcium. As has been seen in studies examining rises in calcium elicited in the DR and the LDT by other peptides such as orexin and ghrelin (Hauberg and Kohlmeier, 2015; Kohlmeier et al., 2004), changes in fluorescence exhibited differences in kinetics that could be categorized into 3 different types: smooth spiker, spiker and plateau responses (Fig 1: A2, B2).

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Using this kinetic profile-based classification, the most common fluorescent response elicited by NPS was of the smooth spiker type, as 60% of the DR responding cells exhibited this response (n = 62/104). The average change in DF/F was 9.1 ± 1.4% (Fig 1: A3). Responses categorized as spikers occurred with the second highest frequency, occurring in 24% of the responding cells (n = 25/104). This response type exhibited an average increase of DF/F of 14.5 ± 2.8% (Fig 1: A3). The plateau type of response was the least common, occurring in 16% of responding DR cells (n = 16/104), which exhibited an average DF/F change of 18.1 ± 3.8% (Fig 1: A3). In order to test the actions of antagonists on NPS actions, we needed to first establish whether effects of NPS were repeatable. In these studies, the second application of NPS was delivered 30 minutes after the first NPS administration. Following this protocol, second applications of NPS elicited responses that were not significantly different in amplitude from first applications in the same cells (n = 25; p = 0.3387; paired Student’s t-test).

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Repeatability of actions of NPS allowed us to conduct within cell studies of the effects of antagonists on NPS actions. For antagonist studies, as the smooth spiker response was the most prevalent, we compared the amplitude of this response type in the presence and absence of the antagonists. We first examined the persistence of the response in presence of blockade of action potentials within the brain slice. Presence of TTX did not significantly alter the response of NPS in eliciting calcium rises, suggesting that voltage-dependent sodium channels are not required for NPS effects on calcium (n = 13; p = 0.95; paired Student’s t-test; Fig 1: A4). As peptides are well known to be capricious, and act beyond their own receptors, we determined whether effects of NPS were specific to activation of the NPS receptor. In the presence of the competitive NPS receptor antagonist, SHA 68, (Okamura et al., 2011, 2008; Patnaik et al., 2013; Ruzza et al., 2012; Thorsell et al., 2013; Trapella et al., 2011), calcium responses to NPS were abolished in cells in which robust responses had been elicited in control conditions, indicating that effects of NPS on eliciting calcium were specific to activation of the NPS receptor (n = 16 ; p < 0.01; paired Student’s t-test; Fig 1: A4, 5).

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NPS induced similar effects on DF/F in Fura 2-AM loaded neurons in the LDT; however, the proportion of cells responding in the LDT was significantly greater than the fraction of cells responding in the DR. NPS elicited a calcium response in 45% of the Fura 2 loaded cells (n = 134/299), which was a significantly larger response rate than that elicited in the DR (p < 0.05; Fisher’s exact test). Similar to responses elicited in the DR, the kinetics of rises in fluorescence in the LDT induced by NPS could be grouped into three categories (Fig 1: B2). In 65% of the responding cells, the smooth spiker response profile was elicited, with an average rise in DF/F of 7.9 ± 0.9% (Fig 1: B3). The second most common response was the spiker response, as it occurred in 24% of the responding cells (average amplitude of the rise: 19 ± 2.2%; Fig 1: B3), with the least common response, the plateau response occurring in 10% of neurons (average amplitude of the rise: 12.8 ± 3.7%; Fig 1: B3). Although significantly more cells responded to NPS in the LDT when compared to the proportion responding in the DR, the distribution of response types and the amplitudes of the responses were not statistically different between nuclei (response type: smooth spikers p = 0.24; spikers p = 0.10; plateau p = 0.48; Fisher’s exact test; amplitude: smooth spikers p = 0.5; spikers: p = 0.2; plateau: p = 0.33; unpaired Student’s t-test).

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As was seen in the DR, following a wash out period of 30 minutes, second applications of NPS were successful in eliciting changes in DF/F in LDT cells that were not statistically different from responses elicited in first applications (n = 31; p = 0.53; paired Student’s t-test). Following establishment of repeatability in the LDT, we then tested the DF/F response to NPS in presence of TTX and compared that to responses elicited in absence of blockade of action potentials within the slice. We found that presence of TTX did not significantly alter the response (n = 16; p = 0.5; paired Student’s t-test; Fig 1: B4). Finally, we examined the ability of the NPS receptor antagonist to inhibit the response to NPS. As seen in the DR, in the presence of SHA 68, NPS failed to elicit any rises in calcium in cells in which NPS application in absence of the receptor antagonist elicited robust rises, (n = 14; p < 0.001; paired Student’s t-test; Fig 1: B4, 5). When taken together, we conclude that NPS has actions on both DR and LDT cells which involve increases in intracellular calcium. These effects were repeatable, and were not occluded by TTX, suggesting actions were independent of action potentials. Further, NPS effects were specific to activation of the NPS receptor. To our knowledge, findings from our Fura 2, calcium imaging

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ACCEPTED MANUSCRIPT experiments provide the first evidence that DR and LDT neurons can be functionally activated by NPS. Based on these encouraging data, we decided to conduct a more investigative examination by using whole cell, patch clamp electrophysiology on individual DR and LDT neurons in order to obtain more information about the cellular effect of NPS in these two brainstem nuclei.

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NPS induces membrane currents in the DR and in the LDT Membrane Effects of NPS on Serotonergic and Non Serotonergic DR Neurons Using whole cell patch clamping, we determined whether NPS had pre and postsynaptic actions on DR and LDT neurons. Voltage clamp recordings confirmed the calcium imaging data by showing that NPS has actions in a large proportion of cells within both nuclei.

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Local application of NPS (10 µM, applied via an adjacent glass pipette) onto DR neurons induced an alteration of the holding current in nearly 70% of the tested neurons (n = 32/46; 12 animals). A deflection from baseline with a response that exhibited kinetics characteristic of peptide responses seen in other studies, which included a gradual rise to a plateau and a slow return to baseline (Kohlmeier et al., 2009), was considered indicative of a response induced by peptide. As a control that responses were peptide mediated, we conducted experiments in which we pressure applied vehicle and in these studies, we never saw deflections from baseline with similar kinetics or latencies, indicating that effects seen were peptide-specific (average amplitude of deflection from baseline: 2.2 ± 0.1 pA, with a latency < 1 min to 2 min, n=14). The most common membrane response was induction of an inward current as NPS induced this membrane response in 72% of the responding cells, (n = 23/32; Fig 2: A1, B). The NPS-induced inward current exhibited an average amplitude of -14.9 ± 2.8 pA (Fig 2: B), and the average latency to the peak of this response was 10.0 ± 1.6 min. In the remaining 28% of responding cells, an outward current with an average amplitude of 5.4 ± 1 pA was induced (n = 9/32; Fig 2: A2, B) with an average latency to peak of 12.6 ± 2.1 min. Immunohistochemistry revealed that NPS-mediated effects occurred in TpH positive cells, as well as TpH negative neurons (Fig 2: D). In our electrophysiological studies, we were able to recover a total of twenty-six Alexa-594 filled cells and process them for immunohistochemistry, which revealed that 46% of the cells that responded to NPS were positive for presence of TpH (n = 7/15). Of the responding cells which were positive for TpH, NPS induced inward currents in 71% of this population (n = 5/7); whereas, outward currents were induced in 29% of these cells (n = 2/7). When taken together, these data indicate that both serotonergic and non-serotonergic neurons in this nucleus respond to NPS, and that induction of inward currents is the most common membrane response. Preliminary experiments demonstrated that effects on the membrane were not repeatable upon second applications of NPS, with only approximately 45% of the membrane response being elicited in second applications, irrespective of elicitation of inward or outward currents in the first application (% response of control: 54.9 ± 5.4%; n = 4; p < 0.01; paired Student’s t-test). When taken in light of our findings that NPS actions on calcium were repeatable using bulk load calcium imaging, our voltage clamp data suggest the possibility that dialysis by the patch solution of intracellular messengers required for mechanisms involved in induction of membrane currents occurred. However, we cannot rule out the possibility that differences in application methods, or effective concentrations of peptide reaching the slice could underlie this difference. Regardless of the explanation, as we wished to

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ACCEPTED MANUSCRIPT proceed with very local applications via an injection pipette, studies with antagonists had to be performed across separate populations.

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To evaluate if the induction of NPS induced membrane currents was dependent on the generation of action potentials within the slice, we tested the effects of NPS during inhibition of voltage-dependent sodium channels by TTX. In the presence of TTX (500 nM), NPS induced membrane responses in 70% of the neurons tested (n = 21/30; 7 animals). Of the responding cells, NPS induced an inward current in 81% of these cells which exhibited an average amplitude of -13.1 ± 2.8 pA (n = 17/21; Fig 2: A3, B); whereas, an outward current with an average amplitude of 4.1 ± 1.6 pA was induced in 19% of those cells (n = 4/21; Fig 2: A4, B). Comparisons between the two populations of cells revealed that the amplitudes of inward currents and outward currents were not statistically different between conditions without TTX when compared to those with TTX present (inward currents: ACSF vs TTX, p=0.65; outward currents: ACSF vs TTX, p=0.51; unpaired Student’s t-test; Fig 2: B). In addition, there were no statistical differences between the proportion of cells responding to NPS when TTX was present when compared to the numbers responding when it was absent, nor were there differences in the distribution of the polarity of the response between the two conditions (total cells responding: ACSF vs TTX, p = 0.5; Cells responding with inward currents: ACSF vs TTX, p = 0.41; cells responding with outward currents: ACSF vs TTX, p = 0.41; Fisher’s exact test).

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As the amplitude of the currents and the polarity distribution were not different in the presence of TTX, this suggested that NPS effects on membrane currents were due to actions on the postsynaptic cell, or to effectors at the presynaptic terminal. To further characterize where NPS effects were occurring, we tested NPS actions on the membrane in a reduced calcium solution, which lowers calcium flux across the pre and postsynaptic membranes, effectively eliminating calcium-dependent synaptic transmission. This solution also eliminates presynaptic release of GABA and glutamate, which was apparent during wash in of the low calcium solution in preliminary recordings by elimination of ongoing synaptic activity. NPS effects on the membrane persisted in low calcium solution. In low calcium solution, NPS elicited membrane currents in the majority of cells (68.8% of the tested neurons, n = 11/16; 3 animals), and the average amplitude of NPS-induced membrane currents was not different from that elicited in normal calcium conditions or TTX free conditions (inward currents: -13.5 ± 2.9 pA, n = 8/11; outward currents: 5.9 ± 1.9 pA, n = 3/11; inward currents: ACSF vs TTX vs Low Ca2+, p = 0.88; outward currents: ACSF vs TTX vs Low Ca2+, p = 0.73; one way ANOVA; Fig 2A5, 6; 2B). Further, neither the proportion of cells responding, or the distribution of response type, was statistically different between the two conditions (total cells responding: ACSF vs TTX vs Low Ca2+, p = 0.99; cells responding with inward currents: ACSF vs TTX vs Low Ca2+, p= 0.74; cells responding with outward currents: ACSF vs TTX vs Low Ca2+, p = 0.74; Fisher’s exact test). These data suggest that membrane responses are due to postsynaptic mechanisms. As responses to NPS in the DR persisted during synaptic blockade, and did not seem to be due to flux of calcium across the membrane, we examined whether intracellular calcium stores were involved in the response, as NPS actions have been shown to include calcium store release (Erdmann et al., 2015). Accordingly, to examine whether SERCA pump stores were necessary for NPS membrane actions, we examined the effect of NPS following depletion of the IP3-mediated stores by CPA. In the presence of CPA (10 µM), NPS still induced membrane currents in the majority of DR cells (70.6%; n = 12/17; 2

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animals). In 66.7% of the responding cells, an inward current was induced (inward: -5.5 ± 1.2 pA, n = 8/12; Fig 2: A7, B); whereas, an outward current was induced in the remaining 33.3% (outward: 2.3 ± 0.3 pA, n = 4/12; Fig 2: A8, B). The numbers of cells responding and the proportion of each response type were not statistically different between CPA absent and CPA present conditions (total cells responding: ACSF vs CPA, p = 0.53; cells responding with inward currents: ACSF vs CPA, p = 0.44; cells responding with outward currents: ACSF vs CPA, p = 0.44; Fisher’s exact test). However, while the numbers of cells responding appeared to be CPA-insensitive, the amplitude of both inward and outward currents induced by NPS in presence of CPA was significantly reduced from amplitudes obtained in control conditions (amplitude ACSF vs CPA, p < 0.05; Mann-Whitney test; Fig 2: B).

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Finally, although current amplitudes were decreased when SERCA pump calcium stores were depleted, they were not eliminated. Therefore, to examine whether calcium sourcing from other intracellular stores was involved in mediating NPS membrane actions, we then examined the ability of NPS to induce membrane effects when applied following depletion of both IP3 and RyR intracellular calcium stores. In presence of TTX, CPA and caffeine (20 mM), NPS failed to induce membrane responses in the DR. In this cocktail, NPS current responses were absent in all of the tested DR neurons (n = 5; 2 animals; p < 0.01; Fisher’s exact test; Fig 2: A9). It is of interest to note that wash-in of the cocktail of CPA and caffeine consistently induced an inward current, which while this was not confirmed, was an effect we attributed to caffeine, as CPA alone did not have this action. When taken together, our data suggest that NPS effects on membrane currents in the DR are postsynaptically mediated, and rely on both IP3 and RyR-sensitive calcium stores.

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Our next step was to identify the nature of the channels and the ions responsible for the change in membrane potential. Accordingly, we applied voltage steps to construct current-voltage (IV) curves in presence of TTX so as to avoid generation of voltage transients due to firing of action potentials. Inward and outward currents induced by NPS were associated with an increase of membrane conductance of approximately 20% and 12%, respectively, which was significant (% response of control; inward: 119.9 ± 3.8%, n = 4/4, 3 animals; p < 0.05; outward: 112.1 ± 3.3%, n = 4/4, 3 animals p < 0.05; paired Student’s t-test; Fig 2: C), suggesting NPS-mediated opening of membrane channels. In those cells in which NPS generated inward currents, IV curves revealed a reversal potential of the NPS current of approximately -58 mV (-57.7 ± 5.8 mV; Fig 2: C). This reversal potential was consistent with activation of a mixed ionic conductance. As a potassium conductance could be involved, we examined the effects of TEA, which is a potassium channel blocker shown to have strong effects on potassium channels in the DR. TEA (10 mM) was found to have no effect on altering the reversal potential associated with the NPS-induced inward current (data not shown). Therefore, we applied a high potassium solution, which revealed an average reversal potential of the NPS-mediated inward current of -23 mV (-23.1 ± 4.5 mV; n = 3; 3 animals; Fig 2: C), which is consistent with activation of a non-specific cation current (NSCC). We also evaluated whether the high potassium solution resulted in a difference in the amplitude of NPS-induced membrane inward currents, and found that in presence of an altered driving force on potassium, the NPS-induced inward current was increased in amplitude in the same cells, which is a finding also consistent with its mediation, in part, by a potassium conductance (% response of control: 265.0 ± 33.0%, n = 3, paired Student’s t-test).

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Examination of IV curves associated with the induction of outward current by NPS revealed a reversal potential of approximately -79 mV (-78.4 ± 0.8 mV; n = 3; 3 animals; Fig 2: C), which is consistent with mediation by a potassium channel. When taken together, we believe our data support the interpretation that outward currents are generated by a potassium conductance, and inward currents are generated by mixed cationic conductances. Although we did not identify the nature of the potassium conductance activated by NPS when inward currents were apparent, we believe our data which showed a shifting of the reversal potential to a more positive one in the presence of a high potassium solution and induction of a significantly greater amplitude membrane current is strongly indicative of involvement of a potassium conductance. Both inward and outward currents rely on intracellular calcium sourcing from a combination of IP3- and RyR-mediated intracellular stores for their activation. While it would be interesting to identify the precise cation conductances underlying NPS membrane effects, it was beyond the scope of the present work. Lastly, to examine the specificity of membrane actions of the peptide at the NPSR, we tested effects of NPS in presence of the competitive NPSR antagonist, SHA 68. As expected, when applied in the presence of SHA 68, NPS failed to induce any effect on the holding potential of DR neurons (n = 3/3; 2 animals; p < 0.05; Fisher’s exact test; Fig 2: A10), which suggests that membrane actions of NPS were specific to activation of the NPS receptor.

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Membrane Effects of NPS on Cholinergic and Non Cholinergic LDT neurons As multiple-cell calcium imaging also indicated that NPS had effects on LDT cells, we then characterized the cellular actions of this peptide within the LDT. Similar to the effects in the DR, NPS induced membrane currents in the majority of LDT neurons (74%; n = 37/50; 17 animals). Within the population of responding cells, inward currents were induced in 73% of the neurons; whereas, in 27% of the cells, NPS induced outward currents (average amplitude of inward current: -22.1 ± 4.5 pA, n = 27/37; Fig: 3A1, B; average amplitude of outward current: 6.9 ± 1.2 pA, n = 10/37; Fig 3:A2, B). The average latency to the peak of the inward current was 9.8 ± 1.9 min, and the average latency to the peak of the outward current was 11.4 ± 1.8 min. In this study, 36 of the patch clamped neurons could be recovered, which revealed that membrane effects were elicited by NPS in both cholinergic and noncholinergic cells (Fig 3: D). Of the recovered LDT cells which responded to NPS, 48% were found to be bNOS+ (n = 10/21). In 80% of the responding bNOS+ cells, NPS induced an inward current (n = 8/10); whereas, an outward current was induced in the remaining 20% (n = 2/10). Similar to findings in the DR, irrespective of elicitation of inward or outward currents, membrane effects of NPS were not repeatable (% response of control; 52.2 ± 3.2%; n = 5; p < 0.001; paired Student’s t-test) and were specific to drug as test applications of vehicle via the injection pipette did not elicit changes in baseline which were similar in kinetics or latencies (average amplitude 2.0 ± 0.1 pA, with a range of latencies from < 1 min to 2 min, n=15). In an interesting contrast to findings in the DR, NPS induced currents were sensitive to presence of TTX. While the numbers of cells responding (67.6%, n = 26/37; 11 animals), and the response type did not differ from non TTX conditions (cells responding: ACSF vs TTX, p = 0.34; proportion of inward and outward currents: 70% and 30%, respectively; ACSF vs TTX, p = 0.48 and p = 0.48; Fisher’s exact test), the average amplitude of both inward and outward currents was significantly reduced by 50% and 46.7%, respectively, when TTX was present (average inward current in TTX: -11.1 ± 2.9 pA; n = 18/26; p < 0.05; Fig 3: A3, B; average outward current in TTX: 3.7 ± 0.4 pA; n = 8/26; p < 0.05;

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ACCEPTED MANUSCRIPT Mann-Whitney test; Fig 3: A4, B). These data indicate that NPS receptors outside the synapse are contributing to membrane responses in the LDT. However, more detailed examination of these receptors was outside the scope of this report and our focus remained on NPS receptors within the presynaptic terminal and the postsynaptic membrane.

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As would be predicted based on our TTX data, low calcium recording conditions also had a significant effect on the membrane currents induced by NPS in LDT neurons. Under these conditions, NPS elicited membrane actions in 65.2% of the LDT cells examined (n = 15/23; 6 animals), which was not a significant difference in response proportion to that obtained in regular calcium conditions (total cells responding: ACSF vs Low Ca2+, p = 0.3; Fisher’s exact test), nor was the distribution of polarity of the membrane response different between the two recording conditions (inward current: 73.3% of the responding cells; outward current: 26.7% of the responding cells; Fisher’s Exact Test for inward and outward: ACSF vs LowCa2+, p = 0.63 and p = 0.63). However, low calcium recording conditions resulted in significantly reduced amplitudes of both the outward and inward currents, when compared to those elicited in control calcium conditions. In low calcium conditions, the inward and outward membrane responses were reduced by 64.7% and 56.5%, respectively when compared to those obtained in control conditions (amplitude of inward current: -7.8 ± 3.4 pA; n = 11/15; p < 0.01; Fig 3: A5, B; amplitude of outward current: 3.0 ± 0.4 pA; n = 4/15; Fig 3: A6, B; p < 0.05; Mann-Whitney test). We compared the numbers of cells responding and the average amplitudes of the responses between TTX and low calcium conditions and found no significant differences (total cells responding: TTX vs Low Ca2+, p = 0.55; proportion of inward and outward: TTX vs Low Ca2+, p = 0.53 and p = 0.53; Fisher’s exact test; amplitude of membrane currents: TTX_inward vs Low Ca2+_inward, p = 0.5; TTX_outward vs Low Ca2+_outward, p = 0.24; unpaired Student’s t-test), which we interpret as further evidence that inhibitory effects of low calcium likely involved inhibition of synaptic transmission.

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Our next step was to determine whether SERCA pump-mediated calcium was involved in the responses in the LDT, as we had found that this intracellular store was involved in DR responses. CPA had an effect on reducing the amplitude of both the inward and outward currents elicited by NPS in LDT cells. (average amplitude of inward currents: -3.8 ± 0.7 pA; n = 12/16; 3 animals; p < 0.0001; Fig 3: A7, B; average amplitude of outward currents: 2.1 ± 0.4 pA; n = 4/16; p < 0.01; Mann-Whitney test; Fig 2: A8, B). While the response amplitude was reduced by presence of CPA, the number of cells responding (84.2%, n = 16/19) was not significantly altered (total cells responding: ACSF vs CPA, p = 0.3; Fisher’s exact test) and the proportion of response types was also not significantly different (inward and outward: 75% and 25%; proportion of inward and outward: ACSF vs CPA, p = 0.58; Fisher’s exact test). Finally, as currents were not entirely abolished by CPA conditions, we evaluated whether RyR channel-mediated calcium stores are also involved in the response, as was seen in the DR. In presence of TTX, CPA and caffeine, NPS failed to induce any change in the membrane holding currents (n = 5/5; 2 animals; p < 0.01; Fisher’s exact test; Fig 3: A9). As in the DR, solutions containing CPA and caffeine induced an inward current, putatively due to effects of caffeine. When taken together, these data suggest that NPS membrane responses in the LDT are complex, comprised of activation of NPS receptors outside the terminal as well as postsynaptic mechanisms, and, those responses occurring postsynaptically require intracellular calcium sourcing from both IP3 and RyR stores.

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We wished to compare the ionic mechanisms underlying the NPS-induced membrane currents between the DR and LDT. Accordingly, we analyzed IV curves from LDT cells in which NPS had a membrane effect in presence of TTX. Currents induced by NPS were accompanied by an increase in membrane conductance, which resulted in an approximate 53% increase from control values for inward currents and 12% for outward currents (% response of control; inward: 152.6 ± 1.9%; n = 4/4; 3 animals; p < 0.001; outward: 112.1 ± 1.4%; n = 4/4; 3 animals; p < 0.01 unpaired Student’s t-test; Fig 3: C). Interestingly, there were differences in the IV relationship between the two nuclei in those cells in which NPS induced an inward current. The reversal potential of the inward current in the LDT was approximately -27 mV (26.9 ± 7.3 mV; n = 4; Fig 3: C), which is likely due to the activation of a NSCC, without a significant contribution from a simultaneously-occurring, potassium-specific conductance, as was believed to have been activated by NPS in the DR. Similar to the findings in the DR, the reversal potential for the outward current was consistent with its being mediated by a K+ conductance (-69 ± 3.5 mV; n = 3; Fig 3: C). Identification of the precise NSCC and K+ channel activated by NPS remains for future studies.

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Finally, specificity of actions of NPS in the LDT at NPS receptors was determined by the failure to elicit any membrane change with NPS in presence of the NPS receptor antagonist. When applied in presence of SHA 68, NPS failed to induce any change in holding current of the membrane (n = 3/3; 2 animals; p < 0.05; Fisher’s exact test; Fig 3: A10), thereby confirming specificity of actions of NPS at the NPS receptor. When taken together, our data indicate that NPS, acting via an NPS receptor, induces membrane currents via a calcium-dependent activation of a NSCC and, an as yet unidentified potassium channel(s), which is similar to membrane effects elicited by this peptide in the DR. However, in contrast to actions in DR cells, inward currents in the LDT did not appear to be coincident with a potassium-mediated current driving the reversal potential more negative. Also in contrast to findings in the DR, a demonstrated TTX and low calcium-sensitive membrane component suggests cellular actions of NPS on LDT cells not only arose from postsynaptic activation of NPS receptors, but also involved a presynaptic contribution distinct from the terminal.

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NPS alters synaptic activity in the DR and LDT NPS enhances the frequency of s/mEPSCs in the DR In nearly 77% of the DR cells recorded, local application of NPS induced a significant decrease in the interval between spontaneous excitatory membrane events (sEPSCs; Ctrl: 284.5 ± 12.4 ms; NPS: 159.2 ± 5.5 ms; n = 10/13; 10 animals; p < 0.05; K-S test; Fig 4: A1, 2, 3, B1). In these cells, the frequency of sEPSCs increased by 109% (normalized value of mean: 208.9 ± 24.1%; n = 10/13; p < 0.01; paired Student’s t-test; Fig 4: B3). An NPS-mediated effect on sEPSCs in the DR could till be detected up to 13 minutes post NPS application in all cells, indicating the effect was persistent (n = 10). In a majority of the responding neurons, the amplitude of the EPSCs was also altered. NPS enhanced the amplitude of sEPSCs by 4.8%, which while a small change, was significant in 66.7% of these cells (Ctrl: 20.2 ± 0.5 pA; NPS: 21.1 ± 0.4 pA; n = 6/9; p < 0.05; K-S test; Fig 4: A3, B2). A role of inhibitory transmission was examined by inclusion of strychnine, GABAzine and CGP 55845 in the ACSF during the recording of a small group of DR cells. In presence of blockers of inhibitory transmission, NPS still induced significant changes in the intervals of EPSCs which were similar to those seen in absence of these blockers (91.7% increase in frequency; n=3/6; 2 animals; p < 0.05; K-S test). In one of these

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cells, NPS significantly increased the average amplitude of the EPSCs by 37.5% (p < 0.05; K-S). In the presence of the GABAA and B and glycine receptor blockers, NPS still elicited outward currents which were not smaller than those seen in absence of blockade of these inhibitory receptors, indicating that generation of outward membrane currents was not dependent on GABA mechanisms (DR Outward current ACSF: 5.4 ± 1.0 pA; n = 9; DR Outward Current Strychnine/GABAzine/CGP: 11.9 ± 1.4; n=5/6; unpaired Student’s t-test; p = 0.002). As expected, presence of the blockers of inhibitory transmission did not prevent elicitation by NPS of membrane inward current (n=1/6). As NPS resulted in an increase in frequency of sEPSCs in postsynaptic DR cells, these data suggest the possibility that NPS acts to increase release of glutamate from presynaptic excitatory synapses in this nucleus.

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The presence of the voltage activated sodium channel blocker TTX (Fig 4: C1, 3, D1) did not prevent the NPS induced decrease in inter-event intervals of the excitatory currents. As expected, the baseline interval of EPSC events in TTX was greater from that in absence of the antagonist, however, in TTX conditions, NPS induced a significant reduction in the interval of mEPSC events in the majority of DR cells (60% of the cells; Ctrl: 497 ± 33 ms; NPS: 271.9 ± 15.7 ms; n = 9/15; 5 animals; p < 0.05; K-S test; Fig 4: D1). NPS increased the frequency of mEPSCs by 109% in the same population of cells (normalized value of mean: 208.7 ± 18.1%; n = 9/15; p < 0.001; paired Student’s t-test; Fig 4: D3). Further, and also similar to non TTX conditions, NPS in presence of synaptic blockade enhanced the amplitude of the EPSCs in a large proportion of the cells (18.3% increase; Ctrl: 26.3 ± 0.7 pA; NPS: 31.1 ± 0.7 pA; n = 4/9; p < 0.05; K-S test; Fig 4; C3, D2). Persistance of NPS actions on excitatory synaptic events in presence of TTX suggests that NPS is acting at targets located within the terminal.

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NPS enhances the frequency of s/mIPSCs in the DR To assess whether NPS also affects inhibitory transmission in the DR, we performed voltage clamp recordings using pipettes filled with a high chloride internal solution, in order to alter the driving force for chloride, and thereby enhance detection of inhibitory postsynaptic potentials (IPSCs) as inward currents. As excitatory transmission also results in inward currents, recordings were performed in presence of the glutamate receptor antagonists, AP5 and DNQX, so as to block NMDA and AMPA receptors, respectively, to silence glutamatergic spontaneous synaptic events.

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NPS did have a significant effect on the occurrence of IPSCs in a large number of examined DR cells (Fig 5: A1, B1). NPS significantly reduced the time between the IPSCs in 56.3% of the recorded neurons (Ctrl: 621.7 ± 44.6 ms; NPS: 294.6 ± 15.9 ms; n=9/16; 4 animals; p < 0.05; K-S test; Fig 5: A1, 3, B1). The frequency of IPSC events increased by 145% (normalized value of mean: 244.5 ± 28.4%; n = 9/16; p < 0.001; paired Student’s t-test; Fig 5: B3), which persisted for over 13 minutes in some cases. In 66.7% of these cells, NPS also enhanced the amplitude by a small, but significant, 1.7% (Ctrl: 41.5 ± 1.7 pA; NPS: 42.2 ± 1.6 pA; n = 6/9; p < 0.01; K-S test; Fig 5: A3, B2). Examination of amplitude distributions showed that IPSCs greater than 200 pA were elicited in NPS conditions, whereas in absence of NPS, IPSCs did not exceed this value (Fig 5: B2). These data suggest that GABAergic transmission can be enhanced by NPS. The amplitudes of the inward current induced in presence of blockade of glutamatergic transmission were not statistically different from the amplitudes of inward current in absence of inhibition of excitatory transmission (DR Inward Current ACSF: 14.9 ± 2.8 pA; n=23; DR Inward Current APV/DNQX: 9.5 ±1.8 pA; n=7; unpaired Student’s t-test; p=0.19)

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Blockade of sodium voltage activated channels with TTX did not prevent the actions of NPS on either the frequency or amplitude of inhibitory events (mIPSCs; Fig 5: C1, 3, D2). In 42.9% of DR neurons tested, a significant decrease of inter-event intervals of mIPSCs was elicited (Ctrl: 505.3 ± 35.9 ms; NPS: 312.9 ± 15.8 ms; n = 6/14; 3 animals; p < 0.05; K-S test; Fig 5: C3, D1), and a 118% increase in mIPSC frequency was noted (normalized value of mean: 217.5 ± 32.1%; n = 6/14; p < 0.05; paired Student’s t-test; Fig 5: C3, D2), which were effects similar to those seen in TTX-free conditions. Furthermore, within the majority of this group of cells (83%), an increase in mIPSC amplitude was elicited by NPS (55.3%; Con: 21.9 ± 0.6 pA; NPS: 34.0 ± 1.0 pA; n = 5/6; p < 0.05; K-S test; Fig 5: C3, D2).

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NPS enhances the frequency of s/mEPSCs in the LDT Investigation of NPS effects on synaptic activity in the LDT revealed some similarities to actions seen on synaptic events in the DR, however, some differences were noted. While NPS resulted in enhancement of the frequency of sEPSC, which was an effect similar to that seen in the DR, effects on amplitude were not as consistent as those elicited in the DR (Fig 6: A1, B2). In 76.5% of the recorded LDT neurons, inter-event intervals were significantly decreased (Ctrl: 170.7 ± 6.6 ms; NPS: 114.7 ± 4.2 ms; n = 13/17; 13 animals; p < 0.05; K-S test; Fig 6: A1, 3, B1), with a 64% increase in the frequency of sEPSCs in these cells (normalized value of mean: 163.5 ± 8.7%; n = 13/17; p < 0.0001; paired Student’s t-test; Fig 6: B3). This effect persisted 13 minutes post NPS application (n=13). However, in 38.5% of these cells, a significant increase of 20% was noted in the amplitude of sEPSCs (Ctrl: 24.5 ± 0.4 pA; NPS: 29.9 ± 0.4 pA; n = 5/13; p < 0.05; K-S test; Fig 6: B2); whereas, in 38.5% of these cells, a significant decrease of 8.4% in the amplitude of sEPSCs was elicited (Ctrl: 35.7 ± 0.5 pA; NPS: 33.4 ± 0.4 pA; n = 5/13; p < 0.05; K-S test; Fig 6: B2). In a small group of cells, examination of effects of NPS on PSC intervals and amplitudes was conducted in presence of blockade of inhibitory transmission. In presence of strychnine, GABAzine and CGP 55845, NPS still induced a significant change in the intervals of sEPSC (Ctrl: 196.6 ± 9.6 ms; NPS: 127.8 ± 4.6 ms; n = 3/6; K-S test, p < 0.0001), which represented a frequency increase of 57.3%. While a significant decrease in amplitude of EPSCs was not noted in this population, an increase in amplitude of sEPSCs was elicited under these conditions in three LDT cells (Ctrl: 17.3 ± 0.3; NPS: 21.8 ± 0.4 pA; n = 3/6; K-S test, p < 0.0001). As was seen in the DR, in presence of blockers of inhibitory transmission NPS still elicited outward currents which were not smaller in amplitude than those recorded in absence of inhibitory synaptic transmission, suggesting GABAergic and glycinergic mechanisms were not required for this membrane action (LDT Outward Current ACSF: 6.8 ± 1.2 pA; n=10; LDT Outward Current Strychnine/GABAzine/CGP: 12.9 ± 3.9 pA; n= 4/6; unpaired Student’s t-test; p = 0.01). As expected, blockers of inhibitory transmission did not affect NPS’s ability to induce inward currents (LDT Inward

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NPS was then applied in presence of TTX to determine whether effects were dependent on action potential generation within the slice (Fig 6: C1). Similar to effects seen in the DR, in the majority of LDT neurons, NPS application in the presence of TTX resulted in a decrease in inter-event intervals (61.5% of the cells, Ctrl: 192.3 ± 10.8 ms; NPS: 127.1 ± 4 ms; n = 8/13; 5 animals; p < 0.05; K-S test; Fig 6: D1), and a 109% increase in frequency of mEPSCs (normalized value of mean: 208.7 ± 28.8%; n = 8/13; p < 0.01; paired Student’s t-test; Fig 6: D3). In presence of TTX, NPS still had actions on the amplitude of excitatory events, which exhibited a similar biphasic profile as seen in non TTX conditions. In 37.5% of the cells, a significant increase of 39% in amplitude of the mEPSCs was elicited (Ctrl: 22.5 ± 0.7 pA; NPS: 32.1 ± 0.9 pA; n = 3/8; p < 0.05; K-S test; Fig 6: D2); whereas, NPS induced a significant decrease in amplitude of mEPSCs in one cell (12%; n = 1/8), which was not a significant reduction in response type from that seen in non TTX conditions (data not shown; p = 0.3359; Fisher’s exact test). When taken together, we conclude that effects of NPS on enhancing synaptic activity were not dependent on voltage-dependent sodium channels, and that NPS has actions on excitatory transmission in the LDT.

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NPS enhances the frequency of s/mIPSCs in the LDT As NPS had actions on GABAergic transmission within the DR, the effects on sIPSCs in the LDT were also examined. Accordingly, NPS was applied in presence of AP5 and DNQX to inhibit excitatory transmission, and LDT neurons were recorded with the chloride-enriched intracellular solution. Findings obtained under these recording conditions were not qualitatively different from those obtained in the DR (Fig 7: A1). In 62.5% of tested LDT neurons, NPS application resulted in a significant decrease of IPSCs inter-event intervals (Ctrl: 644.3 ± 44.7 ms; NPS: 231.3 ± 11.9 ms; n = 10/16; 5 animals; p < 0.05; K-S test; Fig 7: B1). A NPS-mediated 327% increase in frequency of sIPSCs was elicited which included one outlier which increased by 1641% (normalized value of mean: 427.4 ± 142.9%; n = 10/16; p < 0.05; paired Student’s t-test; Fig 7: B3). Effects on frequency could still be detected 13 minutes post NPS application. Further, in the majority of cells responding with NPSmediated frequency increases, a 52% increase in amplitude was elicited, which was significant (70% of cells responded; Ctrl: 24.9 ± 1.1 pA; NPS: 37.7 ± 1.1 pA; n = 7/10; p < 0.05; K-S test; Fig 7: B2). As was seen in the DR, the amplitude of inward current induced by NPS in presence of blockers of glutamatergic transmission was not different than that in presence of AP5 and DNQX (LDT Inward Current ACSF: 22.1 ± 4.5 pA, n = 27; LDT inward current APV/DNQX: 19.4 ± 2.8 pA, n = 9, unpaired Student’s t-test, p=0.2859), suggesting membrane inward currents induced by NPS in the LDT were not dependent on intact excitatory synaptic transmission and further, that they were not reliant on a chloride conductance. Arguing against a role of glutamatergic transmission in NPS-induced outward currents were findings that there was no statistical difference in the amplitudes of outward currents in presence of AP5 and DNQX (4.2 ± 0.6 pA, n=3) from the amplitudes of currents recorded in absence of blockers of excitatory transmission (6.8 ± 1.2 pA; n=10; unpaired Students t-test; p=0.2783). In the presence of TTX, NPS effects on the inhibitory events were not altered from those in non TTX recording conditions (Fig 7: C1, 2). NPS exposure resulted in a decrease in inter-event intervals of

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mIPSCs in LDT cells that was significant (Ctrl: 600.9 ± 50.7 ms; NPS: 267.7 ± 21 ms; n = 5/13; 3 animals; p < 0.05; K-S test; Fig 7: D1). The frequency of mIPSCs was increased by 131% (normalized value of mean: 230.6 ± 28.4%; n = 5/13; p < 0.05; paired Student’s t-test; Fig 7: D3). Although the percentage of cells responding with an increase in mIPSC frequency was reduced from that responding with a frequency increase in absence of TTX (38.5% vs 62.5%, respectively), the proportion of cells responding in TTX with a significant change in frequency was not significantly different than the numbers responding in absence of TTX (Fisher’s exact test; p = 0.53). In 60% of the responding cells, the amplitude of mIPSCs was significantly increased by 47% (Ctrl: 17.1 ± 0.6 pA; NPS: 23.9 ± 1.3 pA; n = 3/5; p < 0.05; K-S test; Fig 7: D2).

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When taken together, our analyses of synaptic events indicates that NPS has significant effects on the frequency of both EPSCs and IPSCs within the DR and the LDT, and that the amplitude of these events is also affected within a subpopulation of these cells. The persistence of these effects in presence of TTX indicates that NPS has actions on presynaptic glutamatergic and GABAergic terminals within both the DR and the LDT. We did note one major difference in the effect of NPS on synaptic activity in that there was the elicitation of an inhibition of amplitude of EPSCs in a subpopulation of cells within the LDT that was absent in the DR. While it did not occur at a high frequency, this effect across nuclei was different, and could be physiologically relevant.

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NPS Induces Calcium Responses in Individual Neurons of the DR and LDT NPS-Induced Rises in Calcium in the DR In our next series of experiments, we conducted electrophysiological recordings concurrent with calcium imaging, in order to monitor effects of NPS on calcium at the single cell level. To conduct calcium imaging, bis-Fura 2 was introduced directly into the cell from the intracellular solution of the pipette following establishment of the patch configuration. As before, NPS was applied via picospritzer positioned nearby the recorded cell. Under these conditions, NPS induced robust changes in DF/F in the majority of recorded DR neurons (87.5% of neurons; n = 14/16; 8 animals). We confirmed that changes in DF/F were not artifact as application of vehicle only rarely induced a brief duration rise in DF/F and when induced, this rise exhibited a relatively-short, average latency of 7.1 ± 1.2 s, n=3 animals). The average rise in NPS-mediated changes in DF/F was 29.6 ± 6.8% and this change arose with an average latency of 123.0 ± 27.7 s. (Fig 8: A1, 5). Similar to effects on membrane currents, NPS-induced rises in calcium were not repeatable, or reversible, despite the fact that often the DF/F did return to baseline values (DF/F of first application: 20.5 ± 3.8%; DF/F of second application: 8.0 ± 1.3%; n = 11; p < 0.05; paired Student’s t-test). As changes in DF/F were repeatable under bulk load conditions, the most parsimonious explanation is that intracellular constituents underlying the NPSmediated rises in calcium were dialyzed and exhausted following the first application. Regardless of the underlying cause, for the next series of studies, we could not conduct within cell comparisons, and had to conduct a population study. Changes in DF/F induced by NPS persisted in presence of TTX. The average change in DF/F induced by NPS in TTX conditions was 25.6 ± 9.0%, which was not significantly different from that induced in control conditions (n = 9/11; 4 animals; p = 0.9; Mann-Whitney test; Fig 8: A2, 5), and the proportion of cells responding in TTX to NPS was also not significantly different (81.8% of neurons; p = 0.36;

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Fisher’s exact test). However, rises in DF/F were significantly altered when a low calcium external solution was utilized. In conditions of low extracellular calcium, the change in DF/F was 33.8% of that obtained in normal calcium conditions, which was a significant reduction (10.0 ± 2.7 % DF/F; 3 animals; p < 0.01; Mann-Whitney test; Fig 8: A3, 5). While the amplitude was significantly affected by low calcium, the proportion of cells responding with changes in fluorescence was not altered under these conditions (76.9% of neurons responded; n = 10/13; p = 0.4; Fisher’s exact test). Depletion of the Ca2+-ATPase (SERCA) intracellular calcium stores via use of CPA resulted in calcium transients, which were significantly reduced to an average DF/F of 4.6 ± 1% (2 animals; p < 0.0001; MannWhitney test; Fig 8: A4, 5). However, the numbers of neurons responding was not significantly different, as 70% of neurons tested responded to NPS with an increase in DF/F despite depletion of the IP3-mediated calcium stores (n = 7/10; p = 0.18; Fisher’s exact test). As NPS-induced some residual change in DF/F in low external calcium, and in CPA conditions, we then monitored NPS calcium responses in presence of CPA and caffeine, which we had seen was effective in eliminating the membrane response. The concomitant depletion of SERCA-mediated and RyR-dependent calcium stores had a profound effect on the kinetic of the calcium rise when compared to that in CPA-only conditions. In CPA-caffeine, in 80% of neurons, NPS induced a short-lived rise in DF/F of 5.6 ± 1.5%, which was not significantly reduced in amplitude from the rise elicited in CPA alone (n = 4/5; 2 animals; p = 0.58; Fisher’s exact test). However, the duration of this transient was much reduced from that seen in CPA-only conditions. In all recordings with CPA-caffeine, the calcium rose to its maximum amplitude and returned to baseline by 10 minutes post-NPS application (Fig 8: A6), whereas, in recordings absent of these compounds, calcium was still elevated at 10 minutes post NPS, and often remained elevated for much longer. Indicating that NPS-induced calcium was specific to activation of NPS receptors were findings that in presence of the NPS receptor antagonist, SHA 68, NPS failed to elicit any rises in calcium (n = 3/3; p < 0.05; Fisher’s exact test; Fig 8: A7).

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NPS-Induced Rises in Calcium in the LDT NPS had similar actions on calcium in LDT cells to those elicited in DR neurons. NPS induced a change in DF/F of 18.8 ± 3% in the majority of neurons examined, with an average latency of 132.8 ± 29.0 s (88.9%; n = 16/18; 9 animals; Fig 8: B1, 5). Pressure injection of vehicle sometimes elicited a rapid change in DF/F which quickly extinguished and returned to baseline (average latency of vehicle induced rise in DF/F 10.3 ± 1.7 s; n=4). Although DF/F often returned to baseline levels, effects on calcium were not reversible or repeatable (DF/F of first application: 18.5 ± 2.4%; DF/F of second application: 7.4 ± 1.9%; n = 12; p < 0.01; paired Student’s t-test). TTX failed to have any effect on the degree of change in DF/F induced by NPS (13.6 ± 1.8%; 5 animals; p = 0.1128; Mann-Whitney test; Fig 8: B2, 5) and on the proportion of cells responding to NPS with a rise in DF/F (85.7%; n = 12/14; p = 0.6; Fisher’s exact test). Reminiscent of findings obtained in the DR, NPS applied in low calcium solutions resulted in a significantly lower increase in DF/F (8.9 ± 1.7%; 4 animals; p < 0.01; MannWhitney test; Fig 8: B3, 5); however, the numbers of cells responding to NPS with a rise in DF/F were not different between low calcium and control conditions (88.9% of neurons; n = 16/18; p = 0.5; Fisher’s exact test). Depletion of IP3-mediated calcium stores did result in a significantly smaller change in DF/F (5.7 ± 0.8% DF/F; 2 animals; p < 0.0001; Mann-Whitney test; Fig 8: B4, 5) without significantly changing the frequency of responding neurons when compared to the proportion

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responding in regular ACSF (81.8%; n = 9/11; p = 0.49; Fisher’s exact test). NPS applied in presence of the depletion of SERCA-pump and the RyR-dependent calcium stores, did not reduce the numbers of cells responding (66.7%; n = 4/6; 2 animals; p = 0.25; Fisher’s exact test). However, while the change in DF/F was not statistically different in amplitude (6.2 ± 1.3%), the change in CPA-caffeine exhibited a much-reduced duration, similar to that seen in DR cells (Fig 8: B6). Finally, in presence of SHA 68, increases in amplitude of DF/F by NPS were not elicited in any of the neurons tested, providing strong data that rises in calcium induced by NPS in the LDT were specific to activation of the NPSR (n = 3/3; 2 animals; p < 0.01; Fisher’s exact test Fig 8: B7). When taken together, our data suggest that activation of the NPSR by NPS results in increases in calcium, which rely in part on IP3and RyR-mediated stores. Persistence of a change in DF/F induced by NPS following depletion of these two stores could reflect involvement of a calcium source independent of these stores; however, elucidation of the source of this small rise in calcium awaits future investigations.

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NPS influences the firing frequency of DR and LDT neurons NPS induced alterations in membrane currents and synaptic events in both DR and LDT neurons, which could result in cellular excitability. Therefore our final question was whether NPS could alter the firing frequency of these cells, which was interesting because if so, this could result in influencing nuclei output. To answer this question, we recorded in current clamp mode and held the cell at a depolarized potential sufficient to induce a train of action potentials (APs; -45 mV), and determined whether NPS application resulted in changes in frequency of firing of APs. In the majority of cells, (72.7%; n = 8/11 3 animals), application of NPS altered the firing frequency of DR neurons. In 62.5% of the responding cells, NPS application significantly decreased the frequency of APs by 10.5% (Ctrl 4.1 ± 0.1 Hz; NPS: 3.7 ± 0.1 Hz; n = 5/8; p < 0.0001; Mann-Whitney test; Fig 9: A1, 2, C1). In the remaining 37.5% of the neurons, NPS application resulted in significantly increasing the frequency of APs by 23.2% (Ctrl: 6.9 ± 0.2 Hz; NPS: 8.5 ± 0.1 Hz; n = 3/8; p < 0.0001; Mann-Whitney test; Fig 9: A3, 4, C1). Contrary to our expectations, significant changes in firing were still elicited in presence of blockers of inhibitory and excitatory transmission. In the presence of a cocktail of AP5, DNQX, GABAzine, CGP 55845 and strychnine, NPS elicited a significant alteration in firing rate in all of the DR cells recorded, with an average decrease of 78.3 ± 8.0 % elicited in 60% of the cells (n=5/9, 3 animals; p = 0.0006) and a 39.5 ± 11.9 % increase in 40% of the DR neurons (n=4/9; 3 animals; p = 0.04; Mann-Whitney test), indicating that alteration of firing rate of DR neurons is not reliant on GABAergic or glutamatergic synaptic transmission. NPS also affected the firing of LDT cells. In the majority of neurons studied (76.9%; n = 10/13; 3 animals), NPS elicited a change in AP frequency that was significant. In 60% of the responding neurons, NPS decreased the frequency of APs by 26.9% following NPS application (Ctrl: 2.6 ± 0.1 Hz; NPS: 1.9 ± 0.1 Hz; n = 6/10; p < 0.0001; Mann-Whitney test; Fig 9: B1, 2, C2). Whereas, in the remaining 40% of responding neurons, NPS increased the frequency of APs such that the discharge rate was 119.4% higher than that of control (Ctrl: 3.6 ± 0.1 Hz; NPS: 7.9 ± 0.3 Hz; n = 4/10; p < 0.0001; Mann-Whitney test; Fig 9: B3, 4, C2). As was seen in the DR, NPS-induced changes in firing rate were not eliminated when blockers of excitatory and inhibitory transmission were included in the ACSF. In the presence of AP5, DNQX, GABAzine, CGP 55845 and strychnine, 60% of the LDT

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To summarize, similar to findings with NPS-induced changes in membrane currents and synaptic events, NPS induced a dual effect on firing in the DR and LDT, with significant responses of both polarities being elicited. There were no differences in the proportions responding between the two nuclei (p = 0.6; Fisher’s exact test), nor was there a nuclei-specific difference in the distribution of response types (decrease: p = 0.6; increase: p = 0.7; Fisher’s exact test). However, there was a difference between the two nuclei regarding the degree to which firing was altered. In those cells in which NPS reduced the firing frequency, the reduction in the LDT was nearly 3 times greater than in the DR (p < 0.01; unpaired Student’s t-test; Fig 9: C3). In those cells in which NPS increased the firing frequency, the NPS-enhancement of firing was nearly 5 fold greater in the LDT, than the increase induced in DR cells (p < 0.0001; unpaired Student’s t-test; Fig 9: C3). While NPS still elicited significant changes in firing in the presence of blockers of GABA and glutamate receptors, the relative difference in magnitude of NPS actions on firing between the two nuclei was no longer significant, suggesting that presynaptic input does play a role in the degree to which NPS can alter firing of DR and LDT neurons (DR vs LDT Decrease firing: p = 0.3037; DR vs LDT Increase firing: p = 0.3327; unpaired Student’s t-test; Fig: C4).

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When taken together, our data show that NPS has actions on the majority of DR and LDT cells sufficient to alter their firing, which were not reliant on presynaptic excitatory or inhibitory input, but which was altered in magnitude by such input. These data suggest that in vivo actions on neurons in these nuclei could influence their output to target structures, thereby participating in behaviors governed by these nuclei, including states of arousal and anxiety.

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As NPS has anxiolytic and arousal effects in behavioral assays when applied in vivo, we hypothesized that this peptide would have cellular effects on neurons within two nuclei that play a role in anxiety and arousal, the DR and LDT. Our hypothesis was supported by the present data showing that application of NPS to mouse brain slices induces robust pre- and postsynaptically-mediated cellular actions on DR and LDT neurons. These responses were irrespective of phenotype as they were elicited in both serotonergic and non-serotonergic cells in the DR and cholinergic and non-cholinergic cells in the LDT. When taken together, our calcium imaging and electrophysiology data provide the first evidence that the NPSR is capable of being functionally activated in these two brainstem nuclei, and accordingly, our findings raise the intriguing possibility that the NPS system plays a role in mediating anxiety and in regulating arousal states, at least in part, via actions on DR and LDT neurons.

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NPS Effects in DR and LDT are Mediated by NPSR and Require Intracellular calcium Stores Using in situ hybridization and immunohistochemistry, presence of the G-protein coupled NPSR has been established within various neuronal groups of the CNS of rodents and humans (Adori et al., 2015; Leonard and Ring, 2011; Xu et al., 2007). Immunoreactivity for the NPSR was detected in the rat DR; however, it was not reported to be present in the LDT (Leonard and Ring, 2011). NPS has been shown in NPSR-expressing cultured cells to bind to its receptor with high affinity (~1 nM), leading to activation of Gs and Gq, resulting in activation of cAMP and phospholipase C (PLC), which is implicated in subsequent robust increases in intracellular calcium (Erdmann et al., 2015; Reinscheid et al., 2005). In our study using bulk load calcium imaging and native tissue, NPS was found to induce rises in calcium in DR and LDT cells, which were abolished by presence of the competitive NPSR antagonist SHA 68, demonstrating for the first time that the NPSR can be functionally activated in the mouse DR and LDT. Within the DR and LDT, effects on inducing rises in calcium were not sensitive to blockade of action potentials, as calcium increases were not significantly different in the same cells when TTX was present from when it was absent, which is a finding similar to that reported in hippocampal cultured neurons (Erdmann et al., 2015). The ability of NPS to induce rises in calcium in neurons of the DR and LDT was confirmed using single cell calcium imaging, however, in this configuration, we were not able to elicit repeat effects of the peptide on eliciting rises in calcium, which was likely due to dialysis of intracellular signaling molecules fundamental to the NPS-induced response. Specificity of NPS actions at the NPSR in mediation of this effect was confirmed, as NPSinduced rises in calcium were never evoked in neurons of the DR or LDT in presence of SHA 68. Further, comparisons of NPS-induced calcium rises across two populations of neurons revealed that TTX did not significantly alter the amplitude in either the DR or the LDT, providing further evidence that rises in calcium were due to activation of NPS receptors located at the presynaptic terminal or postsynaptically. Depletion of intracellular calcium stores by presence of CPA, which provokes release of IP3-sensitive ER calcium reservoirs by inhibition of SERCA pumps, and caffeine, which empties RyR-sensitive calcium stores (Dettbarn and Palade, 1998; Kong et al., 2008; Plenge-Tellechea et al., 1997), resulted in significantly reducing the amplitude of NPS-evoked calcium rises in both nuclei. These findings

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support the interpretation that there is a common intracellular transduction pathway underlying NPSinduced rises in calcium within these two brainstem regions, which involves IP3- and RyR-mediated stores. IP3 receptors (IP3Rs) and RyRs control separate calcium stores and their activation occurs via different mechanisms. IP3Rs rely on the production of IP3 for their opening, whereas RyRs can be activated by high calcium concentrations resulting from IP3-mediated release, leading to a process known as calcium-induced calcium release (CICR). CICR is linked to activation of store-operated calcium entry (SOCE), which can include activation of calcium release activated channels (CRAC) in the cell membrane (For reviews: Endo, 2007; Verkhratsky and Shmigol, 1996). A role of flux across the membrane in NPS-mediated events in DR and LDT neurons is supported by our low extracellular calcium experiments, in which NPS-induced calcium rises were attenuated. In cultured cells, NPS receptor activation was shown to involve CRAC-mediated calcium entry, which was inhibited, but not blocked, by low calcium external solutions, similar to findings obtained in the present study (Erdmann et al., 2015). While we believe our low calcium solution eliminated synaptic participation, as we were not able to test zero calcium conditions, it cannot be ruled out that there was a small contribution to NPS-induced calcium rises from calcium-permeable channels independent of those activated by calcium release mechanisms. In addition, in the presence of CPA and caffeine, we were still able to see an early residual calcium rise, which could be due to incomplete actions of these store-depleting compounds, or to a small flux of calcium across the membrane (Johansson et al., 2009). However, an alternative explanation which is not mutually exclusive, is that NPS recruits calcium stores insensitive to CPA and caffeine. Nevertheless, while it would have been interesting to explore the potential role of alternative internal stores in NPS-evoked calcium, and to identify the molecular source of calcium reliant on flux across the membrane, when taken together, our data do support the interpretation that NPSR activation in DR and LDT neurons leads to IP3- and RyR-mediated intracellular calcium release, which involves flux of calcium across the membrane.

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NPSRs Activation Results in a Potassium and Mixed Cationic Conductance. Patch clamp studies revealed that NPS induced inward or outward currents in the majority of DR and LDT neurons. These effects were not repeatable. The bi-phasic membrane actions of NPS on DR and LDT cells are similar to those reported in rat ventromedial hypothalamic neurons, in which NPS elicited either a depolarization or hyperpolarization, which was progressively reduced in amplitude upon subsequent re-application of NPS (Yoshida et al., 2010). Interestingly, findings with TTX and low calcium solutions suggest differential localization of NPS receptors within the DR and LDT. Synaptic blockade resulted in significant reductions in the amplitude of NPS-induced membrane currents in the LDT, with no effect on the amplitude of current induced in DR cells by this peptide. These data suggest the possibility that NPS receptors contributing to the membrane currents are located both pre and postsynaptically in the LDT and that NPS receptors located on glutamatergic or GABAergic input neurons could be activated; whereas, they could be located solely postsynaptically in the DR. However, the amplitudes of the inward currents were not reduced by glutamate receptor blockers and the outward currents were not reduced in amplitude by inhibition of GABAergic and glycinergic receptors, which strongly suggests that NPS’s effect on inducing outward or inward currents was not dependent on activation of GABAergic/glycinergic, or glutamatergic mechanisms, respectively. Interestingly, outward membrane currents were larger in the presence of blockade of GABA and glycine receptors, which suggests the possibility of involvement of a countering effect on

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Examination of IV curves generated in presence of TTX revealed that the reversal potentials of the NPS-mediated outward currents in DR and LDT neurons were consistent with activation of a potassium conductance. Neuropeptides have been shown to activate potassium channels in other neuronal types. Neuropeptide Y modulates potassium currents in hippocampal, thalamic and ventral basal complex neurons (Paredes et al., 2003; Sun et al., 2001) and galanin and leptin induce outward currents in LC and hippocampal neurons, respectively, which are mediated by activation of potassium conductances (Pieribone et al., 1995; Shanley et al., 2002). Finally, gastrin-releasing peptide (GRP) modulates neuronal activity via an increase of a rectifying potassium conductance in neurons of the suprachiasmatic nucleus (Gamble et al., 2011).

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Examination of IV curves generated in presence of TTX revealed that the reversal potential of the NPSinduced inward current in LDT neurons was approximately -27 mV, which is a reversal potential near that of the non-specific cation channel conductance (NSCC) known to be present in the LDT (Hiruma and Bourque, 1995; Kirkpatrick and Bourque, 1995). The NSCC in LDT neurons has been shown to be involved in other peptide-mediated membrane actions. The depolarizing effect of orexin on LDT cells was found to be due to activation of a NSCC, with a reversal potential between -30 mV and 0 mV (Kohlmeier et al., 2008). Activation of this same current by orexin was also responsible, in part, for the depolarization of neurons of the nucleus tractus solitari. Providing further support that a NSCC current was likely activated in the LDT by NPS, a NSCC component was shown to be involved in the depolarizing effect of NPS on rat hypothalamic neurons (Yoshida et al., 2010) and activation of a NSCC was reported to underlie a NPS-mediated depolarization in LC cells (Jüngling et al., 2012).

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Interestingly, the reversal potential of the NPS-induced current in DR neurons in presence of TTX was found to be more negative than that in LDT cells (approximately -58 mV), which suggested NPSmediated activation of multiple channels, which could include a potassium conductance, as has been shown to be activated by peptides in other cell types (Brown et al., 2002; Kohlmeier et al., 2008; Liu et al., 2002). Consistent with this interpretation, the reversal potential shifted more positively, to -23 mV, when NPS was applied in high external potassium solutions, and the amplitude of the induced inward current was larger in the same cells when the reversal potential for potassium was shifted. Although it was beyond the scope of this report to elucidate the identity of this putative potassium-permeable channel, we did conduct some initial investigations with TEA, which blocks many different potassium channels. TEA was ineffective in blocking any component of the NPS-induced current, suggesting TEA-sensitive channels known to be present in the DR, are not involved (Penington and Kelly, 1993). Further, it was reported that NPS could modulate the Ih current, a hyperpolarization-activated mixed cationic current carried by HCN channels, in cells of the rat amygdala (Zhang et al., 2016). As there is a prominent Ih current in DR and LDT cells evidenced during protocols stepping the holding voltage negative than -80 mV (Biel et al., 2009; Li et al., 2001), we examined the effect of NPS on this current. However, in voltage clamp experiments, we failed to detect any effect of NPS application on the

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The shift to a more positive reversal potential of the NPS-induced inward current in DR cells in presence of high potassium external solutions is consistent with involvement of a role of a NSCC. Interestingly, the NSCC was shown to be activated in DR neurons by orexin, indicating that the NSCC is present in these cells, and can be activated following peptidergic stimulation (Kohlmeier et al., 2008). Although we did not identify the identity of channels mediating the NPS currents, when taken together, our data suggest that NPS-induced outward currents are mediated by a potassium conductance, and inward currents are mediated by a non-specific cation conductance similar to the NSCC in the LDT, with a concurrent, and counterbalancing activation of a potassium conductance in the DR. The elimination of this current by the depletion of intracellular calcium stores by CPA and caffeine suggest that the membrane current(s) underlying the inward and outward currents activated by this peptide are reliant on calcium sourcing from the intracellular stores, which is consistent with other peptide studies which have shown that activation of membrane channels are dependent on rises in intracellular calcium following activation of the PLC/IP3 and AC pathways (Baraban et al., 1985; Beaumont and Zucker, 2000; Li et al., 1992; Numann et al., 1991).

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NPS Influences Synaptic Events NPS not only induced membrane effects on DR and LDT neurons, it also altered synaptic transmission. Specifically, within the DR, NPS increased the frequency of sEPSCs and sIPSCs and this effect was not altered by blockade of action potentials, as the frequency of mEPSCs and mIPSCs was also enhanced by NPS in presence of TTX, suggesting an effect of activation of NPS receptors on presynaptic glutamatergic and GABAergic terminals. In a subset of DR cells, the amplitude of sEPSCs was increased as well as that of mEPSCs, which suggests the possibility that postsynaptic NPS receptors were also affected.

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Effects on synaptic activity in the LDT were subtly different from those in the DR. In the LDT, similar to effects in the DR, the frequency of EPSCs was enhanced by NPS, whereas, unlike findings in the DR, the effect on the amplitude was variable, with some cells exhibiting an increase in EPSC amplitude and other cells responding with a decrease in amplitude. In the presence of blockade of action potentials, an enhancement in frequency of mEPSCs induced by NPS was present and in some cells, an increase in amplitude was noted. A reduction in amplitude of mEPSCs was also elicited in one cell, suggesting this effect was not reliant on action potentials. NPS enhanced the frequency of both sIPSCs and mIPSCs, and in a subpopulation, the amplitude of sIPSCs and mIPSCs was also enhanced. When taken together, these data suggest that NPS receptors are located at both pre and postsynaptic locations on glutamate and GABAergic terminals in the LDT. Our findings which show an effect of NPS on excitatory and inhibitory synaptic transmission in the DR and LDT are similar to those found in neurons of the amygdala in which NPS resulted in significant changes in the frequency and amplitude of both sEPSCs and sIPSCs, as well as mEPSCs and mIPSCs (Jüngling et al., 2008; Meis et al., 2011, 2008; Zhang et al., 2016). Our data suggest that in addition to directly mediated membrane actions, NPS alters the excitability of DR and LDT neurons via modulation of excitatory and inhibitory input directed to these cells, which would vary depending on the distribution of NPS receptors on inputs

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terminating on DR and LDT postsynaptic cells. We expected that NPS effects on synaptic events would underlie the change in firing rate induced by NPS. However, persistence of this effect in presence of blockers of excitatory and inhibitory transmission suggests that NPS alteration of firing was not dependent on GABA, glycine, or glutamate receptors, but rather on a different, as yet unidentified, mechanism. However, NPS effects on synaptic activity did appear to play a role in the magnitude of NPS’s modulation of firing with a substantially greater contribution of synaptic activity to enhancement of firing in the LDT, suggesting heightened actions in this nucleus on firing rate in presence of presynaptic activation. When taken together, our data lead us to suggest that pre and postsynaptic control by NPS confers multiple input targets by which this peptide can influence the excitability and output of neurons within these nuclei.

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NPS Alters the Firing Rate of DR and LDT Neurons NPS was found to significantly influence the firing rate of a subset of DR and LDT neurons, which is similar to findings obtained with NPS in the amygdala in which NPS also influenced firing, however enhancement of firing was the most common response seen in this brain region (Meis et al., 2011, 2008; Zhang et al., 2016). In our study, in two thirds of the cells in which NPS had a significant effect on altering firing, the frequency was reduced; whereas, in the remaining one third, the firing was enhanced. As we did not see any NPS-induced changes of the Ih current, which was the action believed to lead to enhancements in firing in amygdala cells (Zhang et al., 2016), this mechanism did not appear to be involved. Firing changes were not found to rely on presynaptic input, as they persisted in presence of blockade of glutamate and GABA receptors, albeit presence of presynaptic input did play a role in the magnitude of the effect. As we found that NPS had a differential effect in activating a potassium conductance in the DR, which in addition to hyperpolarizing the DR cell, would serve to increase shunting of current, differences in firing across the two nuclei could include, in part, a simple mechanism of reduction in the depolarizing effect of excitatory input. When taken together, although we did not identify the underlying mechanism, the final outcome of NPS actions on cellular firing of DR and LDT cells likely relies on a balance between directly-mediated membrane changes in the postsynaptic cells and indirectly-mediated alterations in pre and postsynaptically-mediated glutamatergic and GABAergic drive. While elucidation of the specific mechanisms underlying the inhibition or enhancement in firing of DR and LDT neurons was beyond the scope of the present study, these data do show that NPS has cellular and synaptic actions on DR and LDT neurons, which depending on the pattern of in vivo synaptic release of peptide, could be expected to influence the functioning of these cells, and their output to target structures. Significance and Conclusions As NPS has been shown to alter the excitability of DR and LDT cells and profoundly impact their neuronal functioning, some consideration should be given of the role this peptide could play in behavior via cellular and synaptic effects on neurons in these two nuclei. In vivo studies with rodents have indicated that the NPS/NPSR system is involved in sleep and wakefulness architecture (Ahnaou and Drinkenburg, 2012; Oishi et al., 2014). This peptide system is likely involved in state control in humans as well as early times to bed and reduction of sleep and rest durations were found in subjects with a SNP (rs324981) located within the agonist binding site of the NPSR1, resulting in a tenfold

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enhanced sensitivity of the receptor to NPS (Gottlieb et al., 2007; Spada et al., 2014). The LDT and DR are critical players in the ascending reticular activating system, which mediates forebrain and cortical waking and arousal, as well as states of sleep (Fuller et al., 2006; Nauta, 1946; Swett and Hobson, 1968). Firing of LDT neurons is high during aroused, wakefulness states, as well as during REM sleep, which is another state characterized by a high degree of cortical activation, with the lowest rates seen during slow wave sleep (Moruzzi and Magoun, 1995). DR neuronal firing also varies across state, with high firing during wakefulness, and progressive reductions in firing across the states of sleep, with the lowest firing during REM sleep (Fornal et al., 1985). Given the intricate role of the LDT and DR in aroused and sleeping states, the effects of NPS on inducing arousal, and of altering sleep architecture could be mediated by state-associated activity of NPS on altering the firing of neurons of the LDT and DR.

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NPS administration in freely behaving animals has also been shown to exert an anxiolytic action, including evoking a reduction of fear (Leonard et al., 2008; Lukas and Neumann, 2012; Pulga et al., 2012; Rizzi et al., 2008; Xu et al., 2004; Zoicas et al., 2016). Via serotonergic projections to the limbic structures involved in modulation of fear and anxiety (Vertes, 1991), the DR has been shown to modulate mood. Effects of DR stimulation on anxiety and fear are conflicting, with many studies showing that activation of the DR can be anxiogenic, whereas, others have shown that DR stimulation results in reducing anxiety and panic (Deakin and Graeff, 1991; Geller and Blum, 1970; Hodges et al., 1987; Kiser et al., 1980; Nogueira and Graeff, 1995; Tye et al., 1977). While the precise role of the DR in modulation of anxiety and fear remains to be determined, the demonstrated inhibitory and excitatory cellular actions of NPS within the DR could be involved in modulating output of this nucleus to limbic regions, and in part, this action could play a role in the anxiolytic effects of this peptide.

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One caveat of our study is that we were not able to recover the majority of neurons recorded and identify their phenotype. We did choose to record from the dorsal portion of the DR and the medial portion of the LDT in which the highest numbers of serotonin and cholinergic neurons can be found, respectively (for review, see Monti 2009; Boucetta and Jones, 2009; Jones et al., 2014). Further, in consideration of the behavioral actions of NPS, we choose to record in the dorsal region of the DR, as it receives projections from several regions of the brain involved in emotional behavior, such as the prefrontal cortex and amydala (Van Bockstaele et al., 2993; Didier-Bazes et al., 1997; Commons et al., 2003) and we recorded in the medial region of the LDT which is known to send projections to arousal centers (Boucetta and Jones, 2009; Jones et al., 2014). Our poor recovery rate of recorded cells which precluded identification of the majority of neurons in this study was likely due to the necessity of very long recording times, which we have seen in other studies can affect our recovery rate (Soni et al., 2015). If we had been able to recover a larger majority of neurons, we could perhaps have made stronger conclusions about the relative presence or absence of NPS receptors on the heterogeneous population of cells in the DR and LDT. For example, it would have been interesting to have determined if a greater relative proportion of NPS effects were exerted in principal cells types in either nuclei, which could inform speculation about the relative role this peptide could play in behaviors controlled by the DR and LDT. In addition, it would have been interesting to record across the subdivisions of the DR and LDT to compare and contrast NPS effects in principal and non principal cells in the different nuclear divisions. Regardless, our data provide the first evidence that NPS could

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be functionally involved in control of activity in these two brain stem nuclei. Therefore, as the DR and LDT are known to control arousal and the sleep/wake cycle as well as to be involved in anxiety and fear, our data showing NPS has cellular actions on neurons in these nuclei provide new knowledge which could assist in targeting the NPS system within these two neuronal groups for pharmacological management of disorders of behavioral state and mood.

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Figure 1 NPS induces changes in fluorescence (DF/F) indicative of rises in calcium in DR and LDT cells as demonstrated by bulk-load calcium imaging. (A1 and B1) Fluorescent images taken under 380 nm wavelength light of DR and LDT cells filled with the AM form of the calcium indicator dye, Fura 2. Three selected regions of interest (ROI, dark circles) are shown as encompassing representative Fura 2 filled cells from which DF/F was monitored in this study (40× magnification, scale bar indicates 20 µm). (A2 and B2) Monitoring changes in fluorescence within these ROIs revealed that bath application of NPS (3 mLs, 100 nM, black bar) resulted in changes in DF/F indicating rises in cytoplasmic calcium. Changes in DF/F exhibited three different kinetics, which based on previous peptide studies in the DR and LDT were designated as smooth spiker, spiker and plateau. (A3 and B3, Histograms) The most common response type was the smooth spiker kinetic, as shown in this histogram of the distribution of response profiles, which also shows the amplitudes of the three different responses, across the population of cells in this study. (A4 and B4) The NPS-induced change in DF/F was not found to depend on generation of action potentials in the slice, as shown in these histograms comparing the amplitudes of responses in control (Ctrl) and TTX conditions across the population of cells. (A5 and B5) Presence of the selective NPSR antagonist, SHA 68, resulted in an attenuation of the NPS response as shown in this single example in control and SHA 68-exposed conditions, and across the population of cells studied, which was a significant effect (A4 and B4). (Two Images in Boxed Panels in A and B) Post-hoc processing of the slices used for calcium imaging in this study was performed, and it was confirmed by use of immunohistochemistry that recordings were conducted in the appropriate nucleus. The DR was identified as shown in this representative slice by presence of clusters of neurons positive for TpH (black box A; 5× magnification, top; 10× magnification, bottom, 488 nm wavelength) indicating presence of serotonergic neurons, which are the principal cell type of this nucleus. The LDT was identified as shown in another representative slice from this study by presence of cells positive for bNOS, a demonstrated reliable marker of cholinergic neurons in the LDT (Two Images in Boxed Panels in B; 5× magnification, top; 10× magnification, bottom, 488 nm wavelength). In all images, AQ indicates the aqueduct. In this and subsequent figures, * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001 and **** denotes p < 0.0001.

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Figure 2 NPS elicits an effect on membrane currents in the majority of DR cells. (A) Representative whole cell recordings from individual DR cells showing DR-mediated induction of inward current (A1) and outward current (A2) in control recording conditions. Actions in inducing inward currents (A3) or outward currents (A4) were not altered by presence of TTX, nor low calcium external solutions (A5, A6). NPS-induced inward (A7) and outward currents (A8) were significantly reduced in presence of CPA, which depletes IP3-mediated calcium stores, suggesting a role of this intracellular calcium store in membrane actions of this peptide. (A9) Effects were abolished in presence of CPA and caffeine, indicating that the RyR-mediated calcium store also participates in elicitation of membrane currents by NPS. Note that CPA and caffeine induces an inward current in DR neurons. (A10) Elimination of membrane effects in presence of SHA 68 demonstrates specificity of membrane actions of NPS at the NPSR. (B) Histograms are shown of the average amplitudes of NPS-induced inward currents (pA; downward going bars) and outward currents (pA; upward going bars) under the different experimental conditions across the population of DR cells. (C, Top Left) IV curves (values for the IV curves

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represent the average of 3 cells) during the NPS-induced inward currents revealed a reversal potential of -57 mV (n = 3), which shifted to -24 mV after applying NPS in a high K+ external solution, indicating a role for a potassium conductance in this NPS membrane effect (Bottom Left). (Top Right) IV curves which are generated as the average values collected from 3 cells during the outward current revealed a reversal potential of -79 mV, suggesting a role of activation of a potassium conductance in this action. (Bottom Right) Normalized responses to voltage steps of ∆ = -20 mV (from -60 mV to -80 mV) in presence of TTX showed that the conductance of DR cells increased during inward currents (left column NPS in TTX; n = 4). Conductance also increased during outward currents consistent with opening of a potassium channel (right column NPS in TTX; n = 4). (D) Immunohistochemistry for TpH revealed that membrane actions of NPS occurred in both serotonergic and non-serotonergic cells. As shown in this representative DR brain slice of two recorded cells (Alexa-594 positive, left panel) which responded to very local application of NPS via an application pipette, one of the responding cells was positive for TpH (top cell), and one was negative, indicating while it was within the boundary of the DR nucleus as defined by TpH positive cells, it was not a principal cell type (bottom cell).

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Figure 3 NPS also has an effect on the membrane of the majority of LDT neurons, however, in contrast to actions in the DR, membrane effects in the LDT were sensitive to synaptic blockade. (A) NPS elicited both inward (A1) and outward currents (A2) in LDT cells in control recording conditions. Currents were attenuated by presence of TTX (A3, A4) and recording solutions low in calcium (A5, A6), suggesting that NPS actions extended beyond the terminal and postsynaptic cell. Intracellular IP3mediated calcium stores also played a role as the NPS membrane response was reduced by presence of CPA (A7, A8). (A9) When caffeine was added to CPA, responses to NPS were eliminated, indicting a role for both IP3 and RyR-mediated intracellular calcium stores in the NPS-elicited membrane response, as was seen in the DR. As also noted in the DR, addition of caffeine to the recording solution elicited an inward current. (A10) Effects were specific to activation of the NPSR, as presence of SHA 68 eliminated any membrane action of NPS. (B, Histograms) Averaged data of the membrane currents induced by NPS (in pA) across the population of LDT cells recorded under the different experimental conditions. (C, Left Panel) Plots of current-voltage relationships revealed that the inward current reversed at -27 mV (averaged values taken from 4 cells), which is close to the reversal potential of the NSCC present in LDT cells. (C, Middle Panel) IV plots of the outward current induced by NPS (averaged values collected from 3 cells) demonstrated a reversal potential of -69 mV. Conductance measurements obtained by analyzing the amount of current elicited by a voltage step of ∆ = -20 mV (from -60 mV to -80 mV) in presence of TTX (C, right panel) showed an increase during the inward currents (left column NPS in TTX; n = 4) consistent with activation of a NSCC. Conductance also increased in 4 other neurons in which an outward current was elicited, consistent with activation of a potassium permeable channel by NPS receptors located either at the terminal or the postsynaptic cell (right column NPS in TTX). (D) Fluorescent images of three cells recorded in this study in the LDT, which were filled with Alexa-594 for post hoc identification (left), and fluorescent image of the same field after processing of the tissue for bNOS, which serves as a marker in the LDT of cholinergic neurons. All 3 of these cells responded to NPS locally applied via a picospritzer. As can be seen in the right panel (bNOS), two of the cells were bNOS positive indicating they were cholinergic (top two cells), whereas, one cell was negative, indicating it was a non-cholinergic LDT neuron (bottom cell). These data indicate that NPS actions occurred across the heterogeneous LDT population.

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Figure 4 NPS increased the frequency of spontaneous and miniature excitatory synaptic events (EPSCs and mEPSCs) in the majority of DR neurons recorded, and in many of these cells, the amplitude was also enhanced. (A, C) Voltage clamp recordings showing synaptic events in absence (A1) and presence of TTX (C1) in two different DR neurons illustrating an increase in excitatory synaptic events (sEPSCs and mEPSCs; downward going deflections) following NPS application under both recording conditions. The number of events (N, in this, and subsequent figures) for these individual cells (30 sec of sampled recording) are shown in A2 and C2 in which it can be seen that the number of events increases by more than twofold. (A3, C3) Cumulative fractions also reveal a significant decrease in the intervals of sEPSCs and mEPSCs as well as an increase in the amplitudes. (B, D) Histograms from the population of cells recorded which revealed a significant decrease in the intervals between sEPSCs and mEPSCs (B1, D1). In some of these cells, the amplitude of EPSCS was also significantly enhanced (B2, D2). The increase in frequency of sEPSCs and mEPSCs in the population of cells is shown in B3 and D3, respectively.

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Figure 5 NPS increased the frequency and amplitude of spontanesous and minature inhibitory synaptic events (sIPSCs and mIPSCs) in the DR. (A, C) Voltage clamp recordings from individual DR cells conducted with high cloride pipettes and in presence of blockade of glutamate receptors to inhibit excitatory transmission (DNQX and AP5) showed that NPS increased the frequency and amplitude of sIPSCs (downward going deflections in these experimental conditions) in control ACSF (A1) and in presence of TTX (mIPSCs, C1). (A2, C2) The plotting of the numbers of events from these individual cells during 30sec of sampled recordings in DNQX and AP5 ACSF revealed more than a twofold increase in the number of IPSCs and mIPSCs after NPS. Further, cumulative fractions showed a significant decrease in the intervals as well as an increase in the amplitudes of sIPSCs (A3) and mIPSCs (C3) after NPS application in these cells. (B, D) Histograms from the population of DR cells responding to NPS with a significant change in IPSCs and mIPSCs indicate the average decrease in the intervals and increase in the amplitudes of IPSCs (B1, B2) and mIPSCs (D1, D2). The average change in frequency across the population of DR cells is shown in B3 and D3 in which it is apparent that the frequency of sIPSCs and mIPSCs more than doubled.

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Figure 6 Similar to actions in the DR, NPS enhanced the frequency of sEPSCs and mEPSCs in neurons of the LDT, however, effects on the amplitude were biphasic. (A) Voltage clamp recordings of two cells in the LDT in which NPS enhanced the frequency of sEPSCs, however in cell (a), the amplitude of sEPSCs was increased, whereas, in cell (b) the amplitude was significantly decreased. (A2) Histograms of the number of events in both cells reflect a significant increase in frequency of sEPSCs. (A3) Cumulative fractions of the intervals (A3, left panel) and amplitudes of sEPSCs (A3, right panel) in both cells show that changes in both these parameters were significant (** and ## in these panels indicates results from cell a, and cell b, respectively). (B) Population analyses showing the significant decrease in sEPSC intervals (B1), and the dual effect on amplitude (B2), as well as the effect on frequency (B3). NPS in presence of TTX also resulted in an increase in the frequency of mEPSCs as can be seen in this representative whole cell recording (C1) and the histogram of the number of events in a 30 s duration of recording (C2). This effect was significant as can be seen in the cumulative fraction plots of the intervals (C3, left panel). Analysis of the population data revealed a significant change in the intervals of mEPSCs in the majority of LDT cells (D1). Increases in amplitude

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Figure 7 NPS also induced an increase in frequency of inhibitory events (sIPSCs) as well as an increase in amplitude in the majority of LDT cells, which was an effect not prevented by TTX (mIPSCs). (A, C) Representative voltage clamp recordings of two LDT cells with high chloride patch pipettes and presence of blockade of glutamatergic excitatory transmission (DNQX and AP5) showing a NPS-mediated enhancement of the frequency and amplitude of sIPSCs (A1) and mIPSCs (C1). The number of sIPSCs (A2) and mIPSC (C2) events in these same cells during 30sec of sampled recording is shown in these histograms in which it can be seen that s/mIPSCs nearly doubled in number following NPS. Cumulative fractions showed a significant decrease in the interval and a significant increase in the amplitude of sIPSCs (A3, Left and Right Panels) and mIPSCs (C3, left and right panels) following NPS. (B and D) Population analyses show a significant effect of NPS on decreasing the intervals (1), and increasing the amplitude (2) and frequency (3) of sIPSCs and mIPSCs.

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Figure 8 NPS elicits rises in calcium in neurons of the DR and LDT. (A, B) Fluorescent images taken at 380 nm wavelength light of a DR and of a LDT neuron filled from the recording pipette with the calcium indicator dye, bis-Fura 2, which alters its spectral properties when calcium is bound (40× magnification, scale bar indicates 50 µm). Changes in fluorescence were elicited by NPS suggesting this peptide induces rises in intracellular calcium (A1, B1). Data are presented as the percentage of relative change in fluorescence (% DF/F) with upward going deflections indicating increases in calcium. NPS-induced rises were present in DR and LDT cells in presence of TTX-containing solutions (A2, B2). However, rises were smaller in low calcium extracellular solutions (A3, B3), and further reduced in presence of CPA (A4, B4), indicating NPS-mediated calcium increases do not rely on action potential generation in the slice, but do involve flux across the membrane and intracellular calcium stores. (A5, B5) Histograms of the change in DF/F induced by NPS in the presence of antagonists and low calcium solutions are shown for the population of cells recorded in the DR and LDT. (A6, B6) The kinetics of the responses to NPS were altered in presence of CPA and caffeine as shown in these single examples of a DF/F recording in a DR and LDT cell. While the maximum amplitude of the response was not significantly different in the presence of this cocktail from that elicited in CPA alone, the maximum change in DF/F occurred at a shorter latency, and the duration of the calcium rise was much more reduced than the longer-lived transient persisting in CPA. Effectiveness of CPA and caffeine in release of IP3- and RyR-mediated calcium stores in neurons of the DR and LDT is indicated in the boxed inserts to the right, in which it is apparent that these compounds elicited rises in DF/F. (A7, B7) The calcium response to NPS was abolished in the presence of the NPSR antagonist, SHA 68, in both the DR and the LDT indicating specificity of the involvement of the NPSR in the calcium inducing effects of the peptide. Figure 9 NPS induces changes in the frequency of firing of action potentials (APs) in the majority of DR and LDT neurons studied. (A1, A3, B1, B3) Shown are current clamp recordings of four representative DR and LDT neurons in this study held at a membrane potential of -45 mV which was

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sufficient to induce firing. Note the decrease in firing of the DR and LDT (A1, B1) following NPS application, and the increase in firing of two other DR and LDT neurons induced by NPS application (A3, B3). The changes in firing induced by NPS were significant, as shown in the histograms depicting the frequency in firing in control conditions and after NPS application for each of the 4 cells (A2, A4, B2 and B4). (C) Paired plots in the DR (1) and LDT (2) illustrating the change in frequency of action potentials (APs) shown as the percentage increase or decrease of control induced by NPS application in a population of DR and LDT neurons in which NPS had a significant effect on the firing frequency. (C3) Summary histograms of the effect of NPS on firing frequency showing that there were differences in the magnitude of the effect on frequency between the DR and LDT. The degree to which firing was reduced in the LDT was nearly 3 fold greater than that in the DR, and the enhancement in firing in the LDT was almost 5 times greater than that induced in the DR by this peptide. (C4) Presence of blockers of GABAA and B and ionotrophic glutamate receptors did not prevent NPS alterations in firing, however, synaptic transmission did play a role in the magnitude of the effect on firing, suggesting that differences in synaptic input in vivo could substantially alter the final output of DR and LDT neurons.

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ACCEPTED MANUSCRIPT References Adori, C., Barde, S., Bogdanovic, N., Uhlén, M., Reinscheid, R.R., Kovacs, G.G., Hökfelt, T., 2015. Neuropeptide S- and Neuropeptide S receptor-expressing neuron populations in the human pons. Frontiers in neuroanatomy 9, 126. doi:10.3389/fnana.2015.00126

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Boucetta S, Cisse Y, Mainville L, Morales M, Jones BE 2014. Discharge profiles across the sleepwaking cycle of identified cholinergic, GABAergic, and glutamatergic neurons in the pontomesencephalic tegmentum of the rat. J Neurosci 34:4708-4727. Clark, S.D., Duangdao, D.M., Schulz, S., Zhang, L., Liu, X., Xu, Y.L., Reinscheid, R.K., 2011. Anatomical characterization of the neuropeptide S system in the mouse brain by in situ hybridization and immunohistochemistry. Journal of Comparative Neurology 519, 1867–1893. doi:10.1002/cne.22606 Commons KG, Connolley KR, Valentino RJ 2003. A neurochemically distinct dorsal raphe-limbic circuit with a potential role in affective disorders. Neuropsychopharmacology 28:206-215.

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NPS induces membrane currents in DR and LDT neurons NPS induces rises in calcium in DR and LDT neurons NPS alters synaptic activity in DR and LDT neurons NPS effects are sufficient to alter firing of DR and LDT neurons